TY - JOUR TI - Making 1X=2X: Studies on dosage compensation in Drosophila AU - Scott, M.J. T2 - NZ BioScience DA - 2001/// PY - 2001/// VL - 9 IS - 4 SP - 23–24 ER - TY - JOUR TI - Progress towards the development of a transgenic strain of the Australian sheep blowfly suitable for a sterile-release program AU - Scott, M.J T2 - NZ BioScience DA - 2001/// PY - 2001/// VL - 9 IS - 2 SP - 11–13 ER - TY - JOUR TI - piggyBac‐mediated transposition in Drosophila melanogaster: an evaluation of the use of constitutive promoters to control transposase gene expression AU - Li, X. AU - Heinrich, J. C. AU - Scott, M. J. T2 - Insect Molecular Biology AB - Transgenic non-Drosophilid insects have been made using insect transposable elements that have a broad host range such as the piggyBac element. However, the success rate is often low. Previous piggyBac helper plasmids have used either the piggyBac or the hsp70 promoter from Drosophila melanogaster to control expression of the transposase gene. Here we show that plasmids with the piggyBac transposase gene regulated by constitutive promoters can be effective 'helpers' for mediating transposition in D. melanogaster. We also present preliminary evidence on the use of an RNA helper that encodes the transposase. Our results suggest that for germ-line transformation of non-Drosophilid insects it may be advantageous to isolate a constitutive promoter from the species of interest to control transposase expression. DA - 2001/10// PY - 2001/10// DO - 10.1046/j.0962-1075.2001.00283.x VL - 10 IS - 5 SP - 447-455 J2 - Insect Molecular Biology LA - en OP - SN - 0962-1075 1365-2583 UR - http://dx.doi.org/10.1046/j.0962-1075.2001.00283.x DB - Crossref ER - TY - JOUR TI - piggyBac-mediated transposition in Drosophila melanogaster: an evaluation of the use of constitutive promoters to control transposase gene expression AU - Li, X AU - Heinrich, JC AU - Scott, MJ T2 - Insect molecular biology DA - 2001/// PY - 2001/// VL - 10 IS - 5 SP - 447-455 ER - TY - JOUR TI - Recruitment of the Male-specific Lethal (MSL) Dosage Compensation Complex to an Autosomally Integrated roXChromatin Entry Site Correlates with an Increased Expression of an Adjacent Reporter Gene in Male Drosophila AU - Henry, Rebecca A. AU - Tews, Birke AU - Li, Xuelei AU - Scott, Maxwell J. T2 - Journal of Biological Chemistry AB - Drosophila dosage compensate (equalize X-linked gene products) by doubling the transcription of most X-linked genes in males. The MSL (male-specific lethal) ribonucleoprotein complex consisting of at least five proteins and two non-coding RNAs (roX1 and roX2) is essential for this transcription response. Recently it has been shown that the X-linked roX1 and roX2 genes each contain at least one chromatin entry site for the MSL complex. In this study we show that insertion of either roX1 or roX2 DNA sequences, upstream of an insulated lacZ reporter gene controlled with the constitutive armadillo promoter (arm-lacZ), results in a significant elevation of expression of lacZ in males. However, full compensation, that is a precise doubling of lacZ expression in males relative to females, was only observed in some lines carrying autosomal insertions of either roX1-arm-lacZ orroX2-arm-lacZ transgenes. Furthermore, we found that a 419-base pair fragment of roX1 that contains an MSL binding site was sufficient to cause a modest elevation of expression oflacZ in males, but this response was significantly less than obtained with a full-length roX1 cDNA. This is the first direct demonstration that insertion of an MSL chromatin entry site on an autosome results in elevated expression in males of genes near the entry site. Drosophila dosage compensate (equalize X-linked gene products) by doubling the transcription of most X-linked genes in males. The MSL (male-specific lethal) ribonucleoprotein complex consisting of at least five proteins and two non-coding RNAs (roX1 and roX2) is essential for this transcription response. Recently it has been shown that the X-linked roX1 and roX2 genes each contain at least one chromatin entry site for the MSL complex. In this study we show that insertion of either roX1 or roX2 DNA sequences, upstream of an insulated lacZ reporter gene controlled with the constitutive armadillo promoter (arm-lacZ), results in a significant elevation of expression of lacZ in males. However, full compensation, that is a precise doubling of lacZ expression in males relative to females, was only observed in some lines carrying autosomal insertions of either roX1-arm-lacZ orroX2-arm-lacZ transgenes. Furthermore, we found that a 419-base pair fragment of roX1 that contains an MSL binding site was sufficient to cause a modest elevation of expression oflacZ in males, but this response was significantly less than obtained with a full-length roX1 cDNA. This is the first direct demonstration that insertion of an MSL chromatin entry site on an autosome results in elevated expression in males of genes near the entry site. male-specific lethal 4′,6-diamidino-2-phenyl-indole base pair(s) kilobase(s) ratio of males to females analysis of variance In the vinegar fly Drosophila melanogaster, males have one X chromosome and females have two. Males dosage compensate by doubling the transcription of most X-linked genes (1Lucchesi J.C. Manning J.E. Adv. Genet. 1987; 24: 371-429Crossref PubMed Scopus (103) Google Scholar). The MSL1 complex is required for this male-specific hypertranscription (2Stuckenholz C. Kageyama Y. Kuroda M.I. Trends Genet. 1999; 15: 454-458Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 3Lucchesi J.C. Curr. Opin. Genet. Dev. 1998; 8: 179-184Crossref PubMed Scopus (93) Google Scholar). The complex is comprised of at least five proteins, MSL1, MSL2, MSL3, MLE, and MOF and two non-coding RNAs, roX1 and roX2. All components of the complex co-localize to hundreds of sites along the male X chromosome (4Bone J.R. Lavender J. Richman R. Palmer M.J. Turner B.M. Kuroda M.I. Genes Dev. 1994; 8: 96-104Crossref PubMed Scopus (257) Google Scholar, 5Franke A. Baker B.S. Mol. Cell. 1999; 4: 117-122Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 6Meller V.H. Gordadze P.R. Park Y. Chu X. Stuckenholz C. Kelley R.L. Kuroda M.I. Curr. Biol. 2000; 10: 136-143Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Two of the proteins have enzymatic activity; MLE is an RNA helicase (7Lee C.G. Chang K.A. Kuroda M.I. Hurwitz J. EMBO J. 1997; 16: 2671-2681Crossref PubMed Scopus (122) Google Scholar) and MOF is a histone acetylase (8Smith E.R. Pannuti A. Gu W. Steurnagel A. Cook R.G. Allis C.D. Lucchesi J.C. Mol. Cell. Biol. 2000; 20: 312-318Crossref PubMed Scopus (255) Google Scholar). In addition, a kinase, JIL-1, preferentially associates with the male X chromosome (9Jin Y. Wang Y. Johansen J. Johansen K.M. J. Cell Biol. 2000; 149: 1005-1010Crossref PubMed Scopus (125) Google Scholar), but it is not known if this protein is essential for dosage compensation. The complex only assembles in males as one of the components, MSL2, is not present in females (10Kelley R.L. Solovyeva I. Lyman L.M. Richman R. Solovyev V. Kuroda M.I. Cell. 1995; 81: 867-877Abstract Full Text PDF PubMed Scopus (252) Google Scholar, 11Bashaw G.J. Baker B.S. Development. 1995; 121: 3245-3258PubMed Google Scholar, 12Zhou S. Yang Y. Scott M.J. Pannuti A. Fehr K.C. Eisen A. Koonin E.V. Fouts D.L. Wrightsman R. Manning J.E. EMBO J. 1995; 14: 2884-2895Crossref PubMed Scopus (145) Google Scholar).The complex is initially targeted to the male X chromosome through binding to 30–40 “high affinity” or “chromatin entry” sites (13Lyman L.M. Copps K. Rastelli L. Kelley R.L. Kuroda M.I. Genetics. 1997; 147: 1743-1753PubMed Google Scholar). The complex is then thought to spread from these sites to other sites on the X chromosome (2Stuckenholz C. Kageyama Y. Kuroda M.I. Trends Genet. 1999; 15: 454-458Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Two of these sites are the X-linkedroX1 and roX2 genes (14Kelley R.L. Meller V.H. Gordadze P.R. Roman G. Davis R.L. Kuroda M.I. Cell. 1999; 98: 513-522Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). That is, the same genes that encode the RNA components of the complex also appear to contain DNA sequences that are recognized by the complex.It has recently been shown that a 217-bp DNA fragment ofroX1 is sufficient to produce an ectopic chromatin entry site when inserted on an autosome (15Kageyama Y. Mengus G. Gilfillan G. Kennedy H.G. Stuckenholz C. Kelley R.L. Becker P.B. Kuroda M.I. EMBO J. 2001; 20: 2236-2245Crossref PubMed Scopus (86) Google Scholar).Previously, we developed an insulated reporter gene system to search for cis-acting X-linked DNA sequences that are required for dosage compensation (16Fitzsimons H.L. Henry R.A. Scott M.J. Genetica. 1999; 105: 215-226Crossref PubMed Scopus (14) Google Scholar). The system consists of the constitutivearmadillo promoter driving expression of the lacZreporter gene and flanked by SCS and SCS′ insulator elements. Seven X-linked DNA fragments totaling 63 kb were tested with the system, but none were found to contain DNA sequences that caused elevated expression of the reporter in males. Here we report that insertion of either roX1 or roX2 DNA sequences upstream of the armadillo promoter results in elevated expression of the lacZ reporter gene in maleDrosophila.DISCUSSIONThere are several lines of evidence that have shown that the MSL complex binds to hundreds of sites along the male X chromosome and causes a 2-fold increase in expression of most X-linked genes in maleDrosophila (1Lucchesi J.C. Manning J.E. Adv. Genet. 1987; 24: 371-429Crossref PubMed Scopus (103) Google Scholar, 2Stuckenholz C. Kageyama Y. Kuroda M.I. Trends Genet. 1999; 15: 454-458Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The most direct evidence for the latter is that males that are homozygous for mutant alleles ofmsl1, msl2, or mle have significantly reduced levels of X-linked but not autosomal enzymes and mlemales have a lower overall rate of X-chromosome transcription (22Belote J.M. Lucchesi J.C. Nature. 1980; 285: 573-575Crossref PubMed Scopus (145) Google Scholar). In this study we have shown that binding of the MSL complex to eitherroX1 or roX2 DNA sequences integrated at autosomal sites correlates with an elevated expression of an adjacentlacZ reporter gene. Indeed, in some of the lines that had either roX1 or roX2 cDNA inserted upstream oflacZ we observed full compensation, that is a doubling of expression in males compared with females carrying the same number of copies of the construct. However, in all of the lines carryingroX1 genomic fragments, a line with a roX2genomic fragment, and some of the lines with roX cDNA upstream of lacZ we observed partial compensation. That is the male/female ratios were significantly greater than one but less than the 2-fold expected if recruitment of the MSL complex leads to a precise doubling of transcription as occurs on the X chromosome. We considered that this partial compensation might be because the MSL complex is only binding to the autosomal roX sequence in a fraction of the cells in a tissue. However, we found that the proportion of nuclei that showed binding to the roX1 BS construct, which shows partial compensation, was the same as compared with a line that showed full compensation (3.7 cDNA line 2). Thus it appears that partial compensation cannot be explained by variability in MSL binding between cells within a tissue. However, we have only analyzed cells from one tissue, third instar larvae salivary glands. We also considered the possibility that one roX sequence might not be sufficient in some autosomal locations to recruit enough MSL complexes to achieve full compensation. If so, a construct that had both roX1 and roX2 cDNAs inserted upstream oflacZ may be more effective at recruiting the complex and would thus show full compensation in most lines. However, the lines tested both showed partial compensation. Thus there must be some other explanation for why some lines show only partial compensation. We think the most likely explanation is that the local chromatin environment at the autosomal integration site influences the level of hyperactivation of the lacZ reporter by the MSL complex. It's known that some autosomal sites are much more permissive to spreading of the complex than others, indicating that the local autosomal chromatin environment can affect at least one function of the MSL complex.We found that lines that were homozygous for the roX1 4.9 genomic-arm-lacZ construct gave significantly lower male/female ratios of β-galactosidase activity than lines that were either heterozygous or homozygous for the roX1 3.7 cDNA-arm-lacZ construct. Because both constructs contain a binding site for the MSL complex, it's not obvious why theroX1 genomic construct should give lower hyperactivation oflacZ in males. One possibility is that this is simply because by chance in all the roX1 genomic lines the transgene integrated into a negative chromosomal environment that was not permissive to full hyperactivation of lacZ in males. However, this would seem unlikely, because all three lines gave similar male/female ratios of β-galactosidase. The genomic construct contains additional DNA sequences from the roX1 gene region compared with the cDNA construct. It's possible that these additional sequences may somehow inhibit hyperactivation of lacZ by the MSL complex. The genomic construct would be expected to produceroX1 RNA, whereas the promoter-less cDNA construct would not. The function of the roX1 RNA in the complex is not known. If the RNA has an inhibitory role, then a localized excess of synthesis of roX1 RNA might result in assembly of an MSL complex that is less effective at elevating expression of the adjacent reporter gene. It has been suggested that in vivo there must be some mechanism for dampening the transcription elevation due to the MOF histone acetylase, because in vitro recombinant MOF is able to increase expression from a nucleosomal template far more than 2-fold (23Akhtar A. Becker P.B. Mol. Cell. 2000; 5: 367-375Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Clearly, further experiments with additional constructs are required to determine the biological significance (if any) of the difference in lacZ hyperactivation between roX1genomic and cDNA constructs.The level of elevation of β-galactosidase activity in males carrying the 419-bp fragment of roX1 that contains the MSL binding site was significantly less than obtained with 3.7-kb roX1cDNA. It's possible that by chance all three roX1 BS lines are inserted into negative chromatin environments that inhibit the MSL complex. We think this is unlikely, because all three lines gave very similar male/female ratios of β-galactosidase activity. Rather it is more likely that binding of the MSL complex to the site inroX1 BS is not sufficient to achieve full compensation. This suggests that other sequences in roX1 3.7 cDNA in addition to the MSL binding site in roX1 BS are required for full roX1 function.In Drosophila, the SCS and SCS′ insulator elements are able to protect a gene from position effects and block a transcription enhancer from acting on a promoter (24Kellum R. Schedl P. Cell. 1991; 64: 941-950Abstract Full Text PDF PubMed Scopus (493) Google Scholar, 25Kellum R. Schedl P. Mol. Cell. Biol. 1992; 12: 2424-2431Crossref PubMed Google Scholar). It has been previously shown that the SCS and SCS′ elements do not block genes from being dosage compensated when inserted onto the X chromosome (16Fitzsimons H.L. Henry R.A. Scott M.J. Genetica. 1999; 105: 215-226Crossref PubMed Scopus (14) Google Scholar, 24Kellum R. Schedl P. Cell. 1991; 64: 941-950Abstract Full Text PDF PubMed Scopus (493) Google Scholar). This suggests that these insulators cannot prevent hypertranscription in males due to the MSL complex. In this study we have tested this directly by placing an SCS′ insulator between a roX2sequence, which contains an MSL binding site, and thelacZ reporter. The SCS′ insulator was unable to block elevation of expression of the reporter in males. Because this insulator can block transcription enhancers, this suggests that the mechanism by which the MSL complex affects transcription may be different from how an enhancer acts on a promoter. In the vinegar fly Drosophila melanogaster, males have one X chromosome and females have two. Males dosage compensate by doubling the transcription of most X-linked genes (1Lucchesi J.C. Manning J.E. Adv. Genet. 1987; 24: 371-429Crossref PubMed Scopus (103) Google Scholar). The MSL1 complex is required for this male-specific hypertranscription (2Stuckenholz C. Kageyama Y. Kuroda M.I. Trends Genet. 1999; 15: 454-458Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 3Lucchesi J.C. Curr. Opin. Genet. Dev. 1998; 8: 179-184Crossref PubMed Scopus (93) Google Scholar). The complex is comprised of at least five proteins, MSL1, MSL2, MSL3, MLE, and MOF and two non-coding RNAs, roX1 and roX2. All components of the complex co-localize to hundreds of sites along the male X chromosome (4Bone J.R. Lavender J. Richman R. Palmer M.J. Turner B.M. Kuroda M.I. Genes Dev. 1994; 8: 96-104Crossref PubMed Scopus (257) Google Scholar, 5Franke A. Baker B.S. Mol. Cell. 1999; 4: 117-122Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 6Meller V.H. Gordadze P.R. Park Y. Chu X. Stuckenholz C. Kelley R.L. Kuroda M.I. Curr. Biol. 2000; 10: 136-143Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Two of the proteins have enzymatic activity; MLE is an RNA helicase (7Lee C.G. Chang K.A. Kuroda M.I. Hurwitz J. EMBO J. 1997; 16: 2671-2681Crossref PubMed Scopus (122) Google Scholar) and MOF is a histone acetylase (8Smith E.R. Pannuti A. Gu W. Steurnagel A. Cook R.G. Allis C.D. Lucchesi J.C. Mol. Cell. Biol. 2000; 20: 312-318Crossref PubMed Scopus (255) Google Scholar). In addition, a kinase, JIL-1, preferentially associates with the male X chromosome (9Jin Y. Wang Y. Johansen J. Johansen K.M. J. Cell Biol. 2000; 149: 1005-1010Crossref PubMed Scopus (125) Google Scholar), but it is not known if this protein is essential for dosage compensation. The complex only assembles in males as one of the components, MSL2, is not present in females (10Kelley R.L. Solovyeva I. Lyman L.M. Richman R. Solovyev V. Kuroda M.I. Cell. 1995; 81: 867-877Abstract Full Text PDF PubMed Scopus (252) Google Scholar, 11Bashaw G.J. Baker B.S. Development. 1995; 121: 3245-3258PubMed Google Scholar, 12Zhou S. Yang Y. Scott M.J. Pannuti A. Fehr K.C. Eisen A. Koonin E.V. Fouts D.L. Wrightsman R. Manning J.E. EMBO J. 1995; 14: 2884-2895Crossref PubMed Scopus (145) Google Scholar). The complex is initially targeted to the male X chromosome through binding to 30–40 “high affinity” or “chromatin entry” sites (13Lyman L.M. Copps K. Rastelli L. Kelley R.L. Kuroda M.I. Genetics. 1997; 147: 1743-1753PubMed Google Scholar). The complex is then thought to spread from these sites to other sites on the X chromosome (2Stuckenholz C. Kageyama Y. Kuroda M.I. Trends Genet. 1999; 15: 454-458Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Two of these sites are the X-linkedroX1 and roX2 genes (14Kelley R.L. Meller V.H. Gordadze P.R. Roman G. Davis R.L. Kuroda M.I. Cell. 1999; 98: 513-522Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). That is, the same genes that encode the RNA components of the complex also appear to contain DNA sequences that are recognized by the complex. It has recently been shown that a 217-bp DNA fragment ofroX1 is sufficient to produce an ectopic chromatin entry site when inserted on an autosome (15Kageyama Y. Mengus G. Gilfillan G. Kennedy H.G. Stuckenholz C. Kelley R.L. Becker P.B. Kuroda M.I. EMBO J. 2001; 20: 2236-2245Crossref PubMed Scopus (86) Google Scholar). Previously, we developed an insulated reporter gene system to search for cis-acting X-linked DNA sequences that are required for dosage compensation (16Fitzsimons H.L. Henry R.A. Scott M.J. Genetica. 1999; 105: 215-226Crossref PubMed Scopus (14) Google Scholar). The system consists of the constitutivearmadillo promoter driving expression of the lacZreporter gene and flanked by SCS and SCS′ insulator elements. Seven X-linked DNA fragments totaling 63 kb were tested with the system, but none were found to contain DNA sequences that caused elevated expression of the reporter in males. Here we report that insertion of either roX1 or roX2 DNA sequences upstream of the armadillo promoter results in elevated expression of the lacZ reporter gene in maleDrosophila. DISCUSSIONThere are several lines of evidence that have shown that the MSL complex binds to hundreds of sites along the male X chromosome and causes a 2-fold increase in expression of most X-linked genes in maleDrosophila (1Lucchesi J.C. Manning J.E. Adv. Genet. 1987; 24: 371-429Crossref PubMed Scopus (103) Google Scholar, 2Stuckenholz C. Kageyama Y. Kuroda M.I. Trends Genet. 1999; 15: 454-458Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The most direct evidence for the latter is that males that are homozygous for mutant alleles ofmsl1, msl2, or mle have significantly reduced levels of X-linked but not autosomal enzymes and mlemales have a lower overall rate of X-chromosome transcription (22Belote J.M. Lucchesi J.C. Nature. 1980; 285: 573-575Crossref PubMed Scopus (145) Google Scholar). In this study we have shown that binding of the MSL complex to eitherroX1 or roX2 DNA sequences integrated at autosomal sites correlates with an elevated expression of an adjacentlacZ reporter gene. Indeed, in some of the lines that had either roX1 or roX2 cDNA inserted upstream oflacZ we observed full compensation, that is a doubling of expression in males compared with females carrying the same number of copies of the construct. However, in all of the lines carryingroX1 genomic fragments, a line with a roX2genomic fragment, and some of the lines with roX cDNA upstream of lacZ we observed partial compensation. That is the male/female ratios were significantly greater than one but less than the 2-fold expected if recruitment of the MSL complex leads to a precise doubling of transcription as occurs on the X chromosome. We considered that this partial compensation might be because the MSL complex is only binding to the autosomal roX sequence in a fraction of the cells in a tissue. However, we found that the proportion of nuclei that showed binding to the roX1 BS construct, which shows partial compensation, was the same as compared with a line that showed full compensation (3.7 cDNA line 2). Thus it appears that partial compensation cannot be explained by variability in MSL binding between cells within a tissue. However, we have only analyzed cells from one tissue, third instar larvae salivary glands. We also considered the possibility that one roX sequence might not be sufficient in some autosomal locations to recruit enough MSL complexes to achieve full compensation. If so, a construct that had both roX1 and roX2 cDNAs inserted upstream oflacZ may be more effective at recruiting the complex and would thus show full compensation in most lines. However, the lines tested both showed partial compensation. Thus there must be some other explanation for why some lines show only partial compensation. We think the most likely explanation is that the local chromatin environment at the autosomal integration site influences the level of hyperactivation of the lacZ reporter by the MSL complex. It's known that some autosomal sites are much more permissive to spreading of the complex than others, indicating that the local autosomal chromatin environment can affect at least one function of the MSL complex.We found that lines that were homozygous for the roX1 4.9 genomic-arm-lacZ construct gave significantly lower male/female ratios of β-galactosidase activity than lines that were either heterozygous or homozygous for the roX1 3.7 cDNA-arm-lacZ construct. Because both constructs contain a binding site for the MSL complex, it's not obvious why theroX1 genomic construct should give lower hyperactivation oflacZ in males. One possibility is that this is simply because by chance in all the roX1 genomic lines the transgene integrated into a negative chromosomal environment that was not permissive to full hyperactivation of lacZ in males. However, this would seem unlikely, because all three lines gave similar male/female ratios of β-galactosidase. The genomic construct contains additional DNA sequences from the roX1 gene region compared with the cDNA construct. It's possible that these additional sequences may somehow inhibit hyperactivation of lacZ by the MSL complex. The genomic construct would be expected to produceroX1 RNA, whereas the promoter-less cDNA construct would not. The function of the roX1 RNA in the complex is not known. If the RNA has an inhibitory role, then a localized excess of synthesis of roX1 RNA might result in assembly of an MSL complex that is less effective at elevating expression of the adjacent reporter gene. It has been suggested that in vivo there must be some mechanism for dampening the transcription elevation due to the MOF histone acetylase, because in vitro recombinant MOF is able to increase expression from a nucleosomal template far more than 2-fold (23Akhtar A. Becker P.B. Mol. Cell. 2000; 5: 367-375Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Clearly, further experiments with additional constructs are required to determine the biological significance (if any) of the difference in lacZ hyperactivation between roX1genomic and cDNA constructs.The level of elevation of β-galactosidase activity in males carrying the 419-bp fragment of roX1 that contains the MSL binding site was significantly less than obtained with 3.7-kb roX1cDNA. It's possible that by chance all three roX1 BS lines are inserted into negative chromatin environments that inhibit the MSL complex. We think this is unlikely, because all three lines gave very similar male/female ratios of β-galactosidase activity. Rather it is more likely that binding of the MSL complex to the site inroX1 BS is not sufficient to achieve full compensation. This suggests that other sequences in roX1 3.7 cDNA in addition to the MSL binding site in roX1 BS are required for full roX1 function.In Drosophila, the SCS and SCS′ insulator elements are able to protect a gene from position effects and block a transcription enhancer from acting on a promoter (24Kellum R. Schedl P. Cell. 1991; 64: 941-950Abstract Full Text PDF PubMed Scopus (493) Google Scholar, 25Kellum R. Schedl P. Mol. Cell. Biol. 1992; 12: 2424-2431Crossref PubMed Google Scholar). It has been previously shown that the SCS and SCS′ elements do not block genes from being dosage compensated when inserted onto the X chromosome (16Fitzsimons H.L. Henry R.A. Scott M.J. Genetica. 1999; 105: 215-226Crossref PubMed Scopus (14) Google Scholar, 24Kellum R. Schedl P. Cell. 1991; 64: 941-950Abstract Full Text PDF PubMed Scopus (493) Google Scholar). This suggests that these insulators cannot prevent hypertranscription in males due to the MSL complex. In this study we have tested this directly by placing an SCS′ insulator between a roX2sequence, which contains an MSL binding site, and thelacZ reporter. The SCS′ insulator was unable to block elevation of expression of the reporter in males. Because this insulator can block transcription enhancers, this suggests that the mechanism by which the MSL complex affects transcription may be different from how an enhancer acts on a promoter. There are several lines of evidence that have shown that the MSL complex binds to hundreds of sites along the male X chromosome and causes a 2-fold increase in expression of most X-linked genes in maleDrosophila (1Lucchesi J.C. Manning J.E. Adv. Genet. 1987; 24: 371-429Crossref PubMed Scopus (103) Google Scholar, 2Stuckenholz C. Kageyama Y. Kuroda M.I. Trends Genet. 1999; 15: 454-458Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The most direct evidence for the latter is that males that are homozygous for mutant alleles ofmsl1, msl2, or mle have significantly reduced levels of X-linked but not autosomal enzymes and mlemales have a lower overall rate of X-chromosome transcription (22Belote J.M. Lucchesi J.C. Nature. 1980; 285: 573-575Crossref PubMed Scopus (145) Google Scholar). In this study we have shown that binding of the MSL complex to eitherroX1 or roX2 DNA sequences integrated at autosomal sites correlates with an elevated expression of an adjacentlacZ reporter gene. Indeed, in some of the lines that had either roX1 or roX2 cDNA inserted upstream oflacZ we observed full compensation, that is a doubling of expression in males compared with females carrying the same number of copies of the construct. However, in all of the lines carryingroX1 genomic fragments, a line with a roX2genomic fragment, and some of the lines with roX cDNA upstream of lacZ we observed partial compensation. That is the male/female ratios were significantly greater than one but less than the 2-fold expected if recruitment of the MSL complex leads to a precise doubling of transcription as occurs on the X chromosome. We considered that this partial compensation might be because the MSL complex is only binding to the autosomal roX sequence in a fraction of the cells in a tissue. However, we found that the proportion of nuclei that showed binding to the roX1 BS construct, which shows partial compensation, was the same as compared with a line that showed full compensation (3.7 cDNA line 2). Thus it appears that partial compensation cannot be explained by variability in MSL binding between cells within a tissue. However, we have only analyzed cells from one tissue, third instar larvae salivary glands. We also considered the possibility that one roX sequence might not be sufficient in some autosomal locations to recruit enough MSL complexes to achieve full compensation. If so, a construct that had both roX1 and roX2 cDNAs inserted upstream oflacZ may be more effective at recruiting the complex and would thus show full compensation in most lines. However, the lines tested both showed partial compensation. Thus there must be some other explanation for why some lines show only partial compensation. We think the most likely explanation is that the local chromatin environment at the autosomal integration site influences the level of hyperactivation of the lacZ reporter by the MSL complex. It's known that some autosomal sites are much more permissive to spreading of the complex than others, indicating that the local autosomal chromatin environment can affect at least one function of the MSL complex. We found that lines that were homozygous for the roX1 4.9 genomic-arm-lacZ construct gave significantly lower male/female ratios of β-galactosidase activity than lines that were either heterozygous or homozygous for the roX1 3.7 cDNA-arm-lacZ construct. Because both constructs contain a binding site for the MSL complex, it's not obvious why theroX1 genomic construct should give lower hyperactivation oflacZ in males. One possibility is that this is simply because by chance in all the roX1 genomic lines the transgene integrated into a negative chromosomal environment that was not permissive to full hyperactivation of lacZ in males. However, this would seem unlikely, because all three lines gave similar male/female ratios of β-galactosidase. The genomic construct contains additional DNA sequences from the roX1 gene region compared with the cDNA construct. It's possible that these additional sequences may somehow inhibit hyperactivation of lacZ by the MSL complex. The genomic construct would be expected to produceroX1 RNA, whereas the promoter-less cDNA construct would not. The function of the roX1 RNA in the complex is not known. If the RNA has an inhibitory role, then a localized excess of synthesis of roX1 RNA might result in assembly of an MSL complex that is less effective at elevating expression of the adjacent reporter gene. It has been suggested that in vivo there must be some mechanism for dampening the transcription elevation due to the MOF histone acetylase, because in vitro recombinant MOF is able to increase expression from a nucleosomal template far more than 2-fold (23Akhtar A. Becker P.B. Mol. Cell. 2000; 5: 367-375Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Clearly, further experiments with additional constructs are required to determine the biological significance (if any) of the difference in lacZ hyperactivation between roX1genomic and cDNA constructs. The level of elevation of β-galactosidase activity in males carrying the 419-bp fragment of roX1 that contains the MSL binding site was significantly less than obtained with 3.7-kb roX1cDNA. It's possible that by chance all three roX1 BS lines are inserted into negative chromatin environments that inhibit the MSL complex. We think this is unlikely, because all three lines gave very similar male/female ratios of β-galactosidase activity. Rather it is more likely that binding of the MSL complex to the site inroX1 BS is not sufficient to achieve full compensation. This suggests that other sequences in roX1 3.7 cDNA in addition to the MSL binding site in roX1 BS are required for full roX1 function. In Drosophila, the SCS and SCS′ insulator elements are able to protect a gene from position effects and block a transcription enhancer from acting on a promoter (24Kellum R. Schedl P. Cell. 1991; 64: 941-950Abstract Full Text PDF PubMed Scopus (493) Google Scholar, 25Kellum R. Schedl P. Mol. Cell. Biol. 1992; 12: 2424-2431Crossref PubMed Google Scholar). It has been previously shown that the SCS and SCS′ elements do not block genes from being dosage compensated when inserted onto the X chromosome (16Fitzsimons H.L. Henry R.A. Scott M.J. Genetica. 1999; 105: 215-226Crossref PubMed Scopus (14) Google Scholar, 24Kellum R. Schedl P. Cell. 1991; 64: 941-950Abstract Full Text PDF PubMed Scopus (493) Google Scholar). This suggests that these insulators cannot prevent hypertranscription in males due to the MSL complex. In this study we have tested this directly by placing an SCS′ insulator between a roX2sequence, which contains an MSL binding site, and thelacZ reporter. The SCS′ insulator was unable to block elevation of expression of the reporter in males. Because this insulator can block transcription enhancers, this suggests that the mechanism by which the MSL complex affects transcription may be different from how an enhancer acts on a promoter. We are very grateful to Mitzi Kuroda for gifts of MSL3 antibody and roX plasmid DNA and for communicating results on the MSL binding site in roX1 prior to publication. We thank Hubert Amrein for the gift of roX1and roX2 cDNA plasmids and Duncan Hedderley for performing the statistical analyses. DA - 2001/8// PY - 2001/8// DO - 10.1074/jbc.M103008200 VL - 276 IS - 34 SP - 31953-31958 J2 - Journal of Biological Chemistry LA - en OP - SN - 0021-9258 UR - http://dx.doi.org/10.1074/jbc.M103008200 DB - Crossref ER - TY - CHAP TI - Quantitative trait loci: statistical methods for mapping their positions AU - Zeng, Z.-B. T2 - Encyclopedia of Genetics A2 - Reeve, Eric C.R. A2 - Black, Isobel PY - 2001/// PB - Fitzroy Dearborn Publishers SN - 9781884964343 ER - TY - JOUR TI - Evolution of the Simulated Data Problem AU - Thomas, Duncan C. AU - Borecki, Ingrid B. AU - Thomson, Glenys AU - Weiss, Ken AU - Almasy, Laura AU - Blangero, John AU - Nielsen, Dahlia AU - Terwilliger, Joseph AU - Zaykin, Dmitri AU - MacCluer, Jean T2 - Genetic Epidemiology AB - The simulated data problem was designed via an interactive process by the Simulation Problem Organizing Committee and the selected data simulators. Based on discussions at the previous Genetic Analysis Workshop, many of the features of previous simulation problems, such as a complex disease, genome scan, and replication, were retained and in addition, a population genetics model was used to generate the simulated genes. We describe the process that was used to structure the problem and summarize the discussions about many of the scientific issues that were considered. DA - 2001/// PY - 2001/// DO - 10.1002/gepi.2001.21.s1.s325 VL - 21 IS - S1 SP - S325-S331 LA - en OP - SN - 0741-0395 UR - http://dx.doi.org/10.1002/gepi.2001.21.s1.s325 DB - Crossref KW - complex diseases KW - linkage disequilibrium KW - pedigree data KW - population genetics KW - simulation ER - TY - JOUR TI - Dwarf8 polymorphisms associate with variation in flowering time AU - Thornsberry, J. M. AU - Goodman, M. M. AU - Doebley, J. AU - Kresovich, S. AU - Nielsen, D. AU - Buckler, E. S. T2 - Nat Genet DA - 2001/// PY - 2001/// VL - 28 IS - 3 SP - 286-289 ER - TY - JOUR TI - Association mapping: where we’ve been, where we’re going AU - Nielsen, D.M. AU - Zaykin, D. T2 - Expert Review of Molecular Diagnostics AB - This paper provides a review of recent work in the area of marker-phenotype association studies, specifically as used for localizing--or mapping--genes affecting a trait of interest. We describe the basis of association mapping and discuss a number of the commonly used techniques. We have also included references to various papers that have evaluated the use of these methods. DA - 2001/// PY - 2001/// DO - 10.1586/14737159.1.3.334 VL - 1 IS - 3 SP - 334-342 ER - TY - JOUR TI - Applying Data Mining Techniques to the Mapping of Complex Disease Genes AU - Czika, W.A. AU - Weir, B.S. AU - Edwards, S.R. AU - Thompson, R.W. AU - Nielsen, D.M. AU - Brocklebank, J.C. AU - Zinkus, C. AU - Martin, E.R. AU - Nobler, K.E. T2 - Genetic Epidemiology AB - The simulated sequence data for the Genetic Analysis Workshop 12 were analyzed using data mining techniques provided by SAS ENTERPRISE MINER Release 4.0 in addition to traditional statistical tests for linkage and association of genetic markers with disease status. We examined two ways of combining these approaches to make use of the covariate data along with the genotypic data. The result of incorporating data mining techniques with more classical methods is an improvement in the analysis, both by correctly classifying the affection status of more individuals and by locating more single nucleotide polymorphisms related to the disease, relative to analyses that use classical methods alone. DA - 2001/// PY - 2001/// DO - 10.1002/gepi.2001.21.s1.s435 VL - 21 IS - S1 SP - S435-S440 LA - en OP - SN - 0741-0395 UR - http://dx.doi.org/10.1002/gepi.2001.21.s1.s435 DB - Crossref ER - TY - JOUR TI - GAW12: Simulated Genome Scan, Sequence, and Family Data for a Common Disease AU - Almasy, Laura AU - Terwilliger, Joseph D. AU - Nielsen, Dahlia AU - Dyer, Thomas D. AU - Zaykin, Dmitri AU - Blangero, John T2 - Genetic Epidemiology AB - The Genetic Analysis Workshop (GAW) 12 simulated data involves a common disease defined by imposing a threshold on a quantitative liability distribution. Associated with the disease are five quantitative risk factors, a quantitative environmental exposure, and a dichotomous environmental variable. Age at disease onset and household membership were also simulated. Genotype data, including 2,855 microsatellites on 22 autosomes, were simulated for 1,497 individuals in 23 families. Phenotype data and sequence data for seven candidate genes were provided for 1,000 of these individuals who were "living" and available for study. Data were simulated for 50 replicate samples in each of two populations, a general population and a population isolate formed from a small group of founders. DA - 2001/// PY - 2001/// DO - 10.1002/gepi.2001.21.s1.s332 VL - 21 IS - S1 SP - S332-S338 LA - en OP - SN - 0741-0395 UR - http://dx.doi.org/10.1002/gepi.2001.21.s1.s332 DB - Crossref KW - population isolate KW - quantitative trait KW - simulation ER - TY - JOUR TI - Association Studies under General Disease Models AU - Nielsen, Dahlia M. AU - Weir, B.S. T2 - Theoretical Population Biology AB - There is great expectation that the levels of association found between genetic markers and disease status will play a role in the location of disease genes. This expectation follows from regarding association as being proportional to linkage disequilibrium and therefore inversely related to recombination value. For disease genes with more than two alleles, the association measure is instead a weighted average of linkage disequilibria, with the weights depending on allele frequencies and genotype susceptibilities at the disease loci. There is no longer a simple relationship, even in expectation, with recombination. We adopt a general framework to examine association mapping methods which helps to clarify the nature of case-control and transmission/disequilibrium-type tests and reveals the relationship between measures of association and coefficients of linkage disequilibrium. In particular, we can show the consequences of additive and nonadditive effects at the trait locus on the behavior of these tests. These concepts have a natural extension to marker haplotypes. The association of two-locus marker haplotypes with disease phenotype depends on a weighted average of three-locus disequilibria (two markers with each disease locus). It is likely that these two-marker analyses will provide additional information in association mapping studies. DA - 2001/11// PY - 2001/11// DO - 10.1006/tpbi.2001.1539 VL - 60 IS - 3 SP - 253-263 J2 - Theoretical Population Biology LA - en OP - SN - 0040-5809 UR - http://dx.doi.org/10.1006/tpbi.2001.1539 DB - Crossref ER - TY - JOUR TI - The ethylene pathway: a paradigm for plant hormone signaling and interaction AU - Alonso, Jose M. AU - Ecker, Joseph R. T2 - Science Signaling DA - 2001/// PY - 2001/// VL - 2001 IS - 70 SP - re1 ER - TY - JOUR TI - Phototropin-related NPL1 controls chloroplast relocation induced by blue light AU - Jarillo, Jose A. AU - Gabrys, Halina AU - Capel, Juan AU - Alonso, Jose M. AU - Ecker, Joseph R. AU - Cashmore, Anthony R. T2 - Nature DA - 2001/// PY - 2001/// VL - 410 IS - 6831 SP - 952-954 ER - TY - JOUR TI - An Arabidopsis circadian clock component interacts with both CRY1 and phyB AU - Jarillo, Jose A. AU - Capel, Juan AU - Tang, Ru-Hang AU - Yang, Hong-Quan AU - Alonso, Jose M. AU - Ecker, Joseph R. AU - Cashmore, Anthony R. T2 - Nature DA - 2001/// PY - 2001/// VL - 410 IS - 6827 SP - 487-490 ER - TY - CHAP TI - QTL Mapping AU - Zeng, Z.-B. T2 - Encyclopedia of Genetics PY - 2001/// DO - 10.1006/rwgn.2001.1441 SP - 1587-1593 OP - PB - Elsevier SN - 9780122270802 UR - http://dx.doi.org/10.1006/rwgn.2001.1441 DB - Crossref ER - TY - JOUR TI - Uncontrolled inbreeding AU - McDaniel, B. T. T2 - Journal of Dairy Science AB - The main objective of this review is provide a general overview of the current inbreeding status in major livestock types and to remind all animal breeders that increasing accuracy and intensity of selection are not the only considerations in a genetic improvement program.A second objective is to set the stage for detailed reviews of the situations in some individual species in this symposium.Intense selection in livestock without careful mating and large effective population size has led to uncontrolled inbreeding in many species.Modern and likely future genetic techniques will probably increase this trend unless steps to alleviate it are taken.Without control of inbreeding, deterioration in lowly heritable but economically important traits such as reproductive efficiency and survival is likely.All breeds and breeders in all species should take steps to reduce uncontrolled inbreeding to maintain genetic variation and improve competitiveness with other food protein sources. DA - 2001/// PY - 2001/// DO - 10.3168/jds.s0022-0302(01)70214-7 VL - 84 SP - 185 ER - TY - JOUR TI - Assessing gene significance from cDNA microarray expression data via mixed models AU - Wolfinger, RD AU - Gibson, G AU - Wolfinger, ED AU - Bennett, L AU - Hamadeh, H AU - Bushel, P AU - Afshari, C AU - Paules, RS T2 - JOURNAL OF COMPUTATIONAL BIOLOGY AB - The determination of a list of differentially expressed genes is a basic objective in many cDNA microarray experiments. We present a statistical approach that allows direct control over the percentage of false positives in such a list and, under certain reasonable assumptions, improves on existing methods with respect to the percentage of false negatives. The method accommodates a wide variety of experimental designs and can simultaneously assess significant differences between multiple types of biological samples. Two interconnected mixed linear models are central to the method and provide a flexible means to properly account for variability both across and within genes. The mixed model also provides a convenient framework for evaluating the statistical power of any particular experimental design and thus enables a researcher to a priori select an appropriate number of replicates. We also suggest some basic graphics for visualizing lists of significant genes. Analyses of published experiments studying human cancer and yeast cells illustrate the results. DA - 2001/// PY - 2001/// DO - 10.1089/106652701753307520 VL - 8 IS - 6 SP - 625-637 SN - 1066-5277 KW - ANOVA KW - cDNA microarray KW - gene expression KW - mixed models KW - statistical significance ER - TY - JOUR TI - The genetic architecture of quantitative traits AU - Mackay, TFC T2 - ANNUAL REVIEW OF GENETICS AB - Phenotypic variation for quantitative traits results from the segregation of alleles at multiple quantitative trait loci (QTL) with effects that are sensitive to the genetic, sexual, and external environments. Major challenges for biology in the post-genome era are to map the molecular polymorphisms responsible for variation in medically, agriculturally, and evolutionarily important complex traits; and to determine their gene frequencies and their homozygous, heterozygous, epistatic, and pleiotropic effects in multiple environments. The ease with which QTL can be mapped to genomic intervals bounded by molecular markers belies the difficulty in matching the QTL to a genetic locus. The latter requires high-resolution recombination or linkage disequilibrium mapping to nominate putative candidate genes, followed by genetic and/or functional complementation and gene expression analyses. Complete genome sequences and improved technologies for polymorphism detection will greatly advance the genetic dissection of quantitative traits in model organisms, which will open avenues for exploration of homologous QTL in related taxa. DA - 2001/// PY - 2001/// DO - 10.1146/annurev.genet.35.102401.090633 VL - 35 SP - 303-339 SN - 1545-2948 KW - quantitative trait loci (QTL) KW - quantitative trait nucleotides (QTN) KW - QTL mapping KW - linkage disequilibrium mapping KW - single nucleotide polymorphisms (SNPs) ER - TY - JOUR TI - Oviposition acceptance and fecundity schedule in the cactophilic sibling species Drosophila buzzatii and D-koepferae on their natural hosts AU - Fanara, J. J. AU - Hasson, E. T2 - Evolution DA - 2001/// PY - 2001/// VL - 55 IS - 12 SP - 2615-2619 ER - TY - JOUR TI - Transgenic tobacco plants expressing the maize Cat2 gene have altered catalase levels that affect plant-pathogen interactions and resistance to oxidative stress AU - Polidoros, AN AU - Mylona, PV AU - Scandalios, JG T2 - TRANSGENIC RESEARCH DA - 2001/12// PY - 2001/12// DO - 10.1023/A:1013027920444 VL - 10 IS - 6 SP - 555-569 SN - 0962-8819 KW - catalase KW - disease resistance KW - gene silencing KW - plant-pathogen interactions KW - reactive oxygen ER - TY - JOUR TI - The contributions of sex, genotype and age to transcriptional variance in Drosophila melanogaster AU - Jin, W AU - Riley, RM AU - Wolfinger, RD AU - White, KP AU - Passador-Gurgel, G AU - Gibson, G T2 - NATURE GENETICS DA - 2001/12// PY - 2001/12// DO - 10.1038/ng766 VL - 29 IS - 4 SP - 389-395 SN - 1061-4036 ER - TY - JOUR TI - Structural organization, regulation, and expression of the chloroplastic superoxide dismutase Sod1 gene in maize AU - Kernodle, SP AU - Scandalios, JG T2 - ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS AB - A cDNA and genomic clone encoding maize chloroplastic Cu/Zn superoxide dismutase Sod1 were isolated. Southern blot analysis indicated little homology between the chloroplastic (Sod1) and the cytosolic (Sod2, Sod4, Sod4A) cDNAs. Sequence analysis of the genomic clone revealed a promoter, transit peptide, and partial coding sequence. The promoter contained several response elements (e.g., for light, cold temperature, xenobiotics) that may be involved in the regulation of the Sod1 gene. Sod1 expression during development and in response to physiological and chemical stressors such as temperature, xenobiotics (paraquat), and light were examined. DA - 2001/7/1/ PY - 2001/7/1/ DO - 10.1006/abbi.2001.2397 VL - 391 IS - 1 SP - 137-147 SN - 1096-0384 KW - antioxidants KW - superoxide dismutase KW - gene expression KW - maize KW - oxidative stress KW - cis-elements KW - chloroplast SOD ER - TY - JOUR TI - Preliminary interspecific genetic maps of the Populus genome constructed from RAPD markers AU - Yin, TM AU - Huang, MR AU - Wang, MX AU - Zhu, LH AU - Zeng, ZB AU - Wu, RL T2 - GENOME DA - 2001/8// PY - 2001/8// DO - 10.1139/gen-44-4-602 VL - 44 IS - 4 SP - 602-609 SN - 0831-2796 KW - interspecific hybrids KW - linkage map KW - poplar KW - pseudo-testcross mapping strategy ER - TY - JOUR TI - Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR AU - Ralph, J AU - Lapierre, C AU - Marita, JM AU - Kim, H AU - Lu, FC AU - Hatfield, RD AU - Ralph, S AU - Chapple, C AU - Franke, R AU - Hemm, MR AU - Van Doorsselaere, J AU - Sederoff, RR AU - DM O'Malley, AU - Scott, JT AU - MacKay, JJ AU - Yahiaoui, N AU - Boudet, AM AU - Pean, M AU - Pilate, G AU - Jouanin, L AU - Boerjan, W T2 - PHYTOCHEMISTRY AB - Studying lignin-biosynthetic-pathway mutants and transgenics provides insights into plant responses to perturbations of the lignification system, and enhances our understanding of normal lignification. When enzymes late in the pathway are downregulated, significant changes in the composition and structure of lignin may result. NMR spectroscopy provides powerful diagnostic tools for elucidating structures in the difficult lignin polymer, hinting at the chemical and biochemical changes that have occurred. COMT (caffeic acid O-methyl transferase) downregulation in poplar results in the incorporation of 5-hydroxyconiferyl alcohol into lignins via typical radical coupling reactions, but post-coupling quinone methide internal trapping reactions produce novel benzodioxane units in the lignin. CAD (cinnamyl alcohol dehydrogenase) downregulation results in the incorporation of the hydroxycinnamyl aldehyde monolignol precursors intimately into the polymer. Sinapyl aldehyde cross-couples 8-O-4 with both guaiacyl and syringyl units in the growing polymer, whereas coniferyl aldehyde cross-couples 8-O-4 only with syringyl units, reflecting simple chemical cross-coupling propensities. The incorporation of hydroxycinnamyl aldehyde and 5-hydroxyconiferyl alcohol monomers indicates that these monolignol intermediates are secreted to the cell wall for lignification. The recognition that novel units can incorporate into lignins portends significantly expanded opportunities for engineering the composition and consequent properties of lignin for improved utilization of valuable plant resources. DA - 2001/7// PY - 2001/7// DO - 10.1016/S0031-9422(01)00109-1 VL - 57 IS - 6 SP - 993-1003 SN - 0031-9422 KW - NMR KW - transgenic KW - mutant KW - O-methyltransferase KW - monolignol KW - coniferyl aldehyde KW - sinapyl aldehyde KW - 5-hydroxyconiferyl alcohol ER - TY - JOUR TI - Efforts to initiate construction of a disease resistance package on a designer chromosome in tobacco AU - Lewis, RS AU - Wernsman, EA T2 - CROP SCIENCE AB - Gene cloning and transformation can be used to circumvent linkage drag effects that can plague conventional interspecific gene transfers. These techniques can also be used to create desirable genetic linkages. Use of Nicotiana glutinosa L. N ‐gene mediated TMV (tobacco mosaic virus) resistance in flue‐cured tobacco, N tabacum L., has been limited due to linkage drag effects. Transformation was used to introduce the cloned N ‐gene into NC152, a chromosome addition line possessing a chromosome pair from N africana. This chromosome has been proposed to be used as a “designer chromosome” into which numerous transgenes could be inserted to form a desirable linkage package. The system could be used to shuttle a large number of transgenes from genotype to genotype. One hundred thirty‐six primary transformants possessing the N transgene were produced and hybridized with TMV‐susceptible ‘Petite Havana.’ These may serve as valuable TMV‐resistant breeding materials. For each independent transformant, BC 1 F 1 families which segregated for TMV resistance and the addition chromosome were generated. Data from cosegregation, transmission, and molecular analyses were used to conclude that one transformant possessed an insertion of the N ‐gene in the addition chromosome. By inserting N in the chromosome, we initiated construction of a disease resistance package by linking the TMV resistance gene with a potyvirus resistance gene(s) native to the chromosome. Occasional loss of the transgene, however, may be evidence of previously undetected interchromosomal recombination, and may have implications for use of this system in cultivar development. DA - 2001/// PY - 2001/// DO - 10.2135/cropsci2001.4151420x VL - 41 IS - 5 SP - 1420-1427 SN - 0011-183X ER - TY - JOUR TI - Dwarf8 polymorphisms associate with variation in flowering time AU - Thornsberry, Jeffry M. AU - Goodman, Major M. AU - Doebley, John AU - Kresovich, Stephen AU - Nielsen, Dahlia AU - Buckler, Edward S. T2 - Nature Genetics DA - 2001/7// PY - 2001/7// DO - 10.1038/90135 VL - 28 IS - 3 SP - 286-289 J2 - Nat Genet LA - en OP - SN - 1061-4036 1546-1718 UR - http://dx.doi.org/10.1038/90135 DB - Crossref ER - TY - JOUR TI - Accelerated regulatory gene evolution in an adaptive radiation AU - Barrier, M AU - Robichaux, RH AU - Purugganan, MD T2 - PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA AB - The disparity between rates of morphological and molecular evolution remains a key paradox in evolutionary genetics. A proposed resolution to this paradox has been the conjecture that morphological evolution proceeds via diversification in regulatory loci, and that phenotypic evolution may correlate better with regulatory gene divergence. This conjecture can be tested by examining rates of regulatory gene evolution in species that display rapid morphological diversification within adaptive radiations. We have isolated homologues to the Arabidopsis APETALA3 (ASAP3/TM6) and APETALA1 (ASAP1) floral regulatory genes and the CHLOROPHYLL A/B BINDING PROTEIN9 (ASCAB9) photosynthetic structural gene from species in the Hawaiian silversword alliance, a premier example of plant adaptive radiation. We have compared rates of regulatory and structural gene evolution in the Hawaiian species to those in related species of North American tarweeds. Molecular evolutionary analyses indicate significant increases in nonsynonymous relative to synonymous nucleotide substitution rates in the ASAP3/TM6 and ASAP1 regulatory genes in the rapidly evolving Hawaiian species. By contrast, no general increase is evident in neutral mutation rates for these loci in the Hawaiian species. An increase in nonsynonymous relative to synonymous nucleotide substitution rate is also evident in the ASCAB9 structural gene in the Hawaiian species, but not to the extent displayed in the regulatory loci. The significantly accelerated rates of regulatory gene evolution in the Hawaiian species may reflect the influence of allopolyploidy or of selection and adaptive divergence. The analyses suggest that accelerated rates of regulatory gene evolution may accompany rapid morphological diversification in adaptive radiations. DA - 2001/8/28/ PY - 2001/8/28/ DO - 10.1073/pnas.181257698 VL - 98 IS - 18 SP - 10208-10213 SN - 0027-8424 KW - floral regulatory loci KW - MADS-box KW - CAB9 KW - Hawaiian silversword alliance ER - TY - JOUR TI - The Ca2+ status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants AU - Persson, S AU - Wyatt, SE AU - Love, J AU - Thompson, WF AU - Robertson, D AU - Boss, WF T2 - PLANT PHYSIOLOGY AB - Abstract To investigate the endoplasmic reticulum (ER) Ca2+ stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca2+-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca2+uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent 45Ca2+accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca2+ ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of45Ca2+ released, and a 2- to 3-fold increase in the amount of 45Ca2+ retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca2+ pool. In addition, CRTtransgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca2+-containing medium to Ca2+-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca2+ stores and thereby enhances the survival of plants grown in low Ca2+ medium. DA - 2001/7// PY - 2001/7// DO - 10.1104/pp.126.3.1092 VL - 126 IS - 3 SP - 1092-1104 SN - 1532-2548 ER - TY - JOUR TI - Ribosome-inactivating proteins: A plant perspective AU - Nielsen, K AU - Boston, RS T2 - ANNUAL REVIEW OF PLANT PHYSIOLOGY AND PLANT MOLECULAR BIOLOGY AB - Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate the universally conserved alpha-sarcin loop of large rRNAs. This depurination inactivates the ribosome, thereby blocking its further participation in protein synthesis. RIPs are widely distributed among different plant genera and within a variety of different tissues. Recent work has shown that enzymatic activity of at least some RIPs is not limited to site-specific action on the large rRNAs of ribosomes but extends to depurination and even nucleic acid scission of other targets. Characterization of the physiological effects of RIPs on mammalian cells has implicated apoptotic pathways. For plants, RIPs have been linked to defense by antiviral, antifungal, and insecticidal properties demonstrated in vitro and in transgenic plants. How these effects are brought about, however, remains unresolved. At the least, these results, together with others summarized here, point to a complex biological role. With genetic, genomic, molecular, and structural tools now available for integrating different experimental approaches, we should further our understanding of these multifunctional proteins and their physiological functions in plants. DA - 2001/// PY - 2001/// DO - 10.1146/annurev.arplant.52.1.785 VL - 52 SP - 785-816 SN - 1040-2519 KW - RIP KW - protein synthesis inhibitor KW - plant toxins KW - cytotoxicity KW - 28S ribosomal RNA ER - TY - JOUR TI - Proliferating cell nuclear antigen transcription is repressed through an E2F consensus element and activated by geminivirus infection in mature leaves AU - Egelkrout, EM AU - Robertson, D AU - Hanley-Bowdoin, L T2 - PLANT CELL AB - The geminivirus tomato golden mosaic virus (TGMV) amplifies its DNA genome in differentiated plant cells that lack detectable levels of DNA replication enzymes. Earlier studies showed that TGMV induces the accumulation of proliferating cell nuclear antigen (PCNA), the processivity factor for DNA polymerase delta, in mature cells of Nicotiana benthamiana. We sought to determine if PCNA protein accumulation reflects transcriptional activation of the host gene. RNA gel blot analysis detected an approximately 1200-nucleotide PCNA transcript in young leaves. The same RNA was found in mature leaves of infected but not healthy plants. Reporter gene analysis showed that a 633-bp promoter fragment of the N. benthamiana PCNA gene supports high levels of expression in cultured cells and in young but not mature leaves of healthy transgenic plants. In contrast, PCNA promoter activity was detected in both young and mature leaves of TGMV-infected plants. Developmental studies established a strong relationship between symptom severity, viral DNA accumulation, PCNA promoter activity, and endogenous PCNA mRNA levels. Mutation of an E2F consensus element in the PCNA promoter had no effect on its activity in young leaves but increased transcription in healthy mature leaves. Unlike the wild-type PCNA promoter, TGMV infection had no detectable effect on the activity of the mutant E2F promoter. Together, these results demonstrate that geminivirus infection induces the accumulation of a host replication factor by activating transcription of its gene in mature tissues, most likely by overcoming E2F-mediated repression. DA - 2001/6// PY - 2001/6// DO - 10.1105/tpc.13.6.1437 VL - 13 IS - 6 SP - 1437-1452 SN - 1531-298X ER - TY - PAT TI - Method for reducing expression variability of transgenes in plant cells AU - Thompson, W. F. C2 - 2001/// DA - 2001/// PY - 2001/// ER - TY - JOUR TI - Induction of lipid metabolic enzymes during the endoplasmic reticulum stress response in plants AU - Shank, KJ AU - Su, P AU - Brglez, I AU - Boss, WF AU - Dewey, RE AU - Boston, RS T2 - PLANT PHYSIOLOGY AB - Abstract The endoplasmic reticulum (ER) stress response is a signal transduction pathway activated by the perturbation of normal ER metabolism. We used the maize (Zea mays)floury-2 (fl2) mutant and soybean (Glycine max) suspension cultures treated with tunicamycin (Tm) to investigate the ER stress response as it relates to phospholipid metabolism in plants. Four key phospholipid biosynthetic enzymes, including DG kinase and phosphatidylinositol (PI) 4-phosphate 5-kinase were up-regulated in the fl2 mutant, specifically in protein body fractions where the mutation has its greatest effect. The third up-regulated enzyme, choline-phosphate cytidylyltransferase, was regulated by fl2 gene dosage and developmental signals. Elevated accumulation of the fourth enzyme, PI 4-kinase, was observed in the fl2 endosperm and soybean cells treated with Tm. The activation of these phospholipid biosynthetic enzymes was accompanied by alterations in membrane lipid synthesis and accumulation. The fl2 mutant exhibited increased PI content in protein body membranes at 18 d after pollination and more than 3-fold higher triacylglycerol accumulation in the endosperm by 36 d after pollination. Incorporation of radiolabeled acetate into phospholipids in soybean culture cells increased by about 30% with Tm treatment. The coordinated regulation of ER stress related proteins and multiple components of phospholipid biosynthesis is consistent with signaling through a common pathway. We postulate that the plant ER stress response has an important role in general plant metabolism, and more specifically in integrating the synthesis of protein and lipid reserves to allow proper seed formation. DA - 2001/5// PY - 2001/5// DO - 10.1104/pp.126.1.267 VL - 126 IS - 1 SP - 267-277 SN - 0032-0889 ER - TY - PCOMM TI - Increasing loop domain size does not diminish effects of matrix attachment regions on transgene expression in tobacco cells in culture AU - Mendu, N AU - Massel, M AU - Spiker, S AB - It is now widely held that the chromatin of eukaryotic organisms is organized into loop domains in which chromatin fibers are attached to the nuclear matrix by specific interactions of nuclear matrix proteins with DNA sequences called matrix attachment regions (MARs) [1, 2]. Several MARs have been isolated and incorporated into constructs used for transformation. Work with animal cell culture systems has consistently shown much higher average levels of reporter gene expression in cell lines in which reporter genes are flanked with cloned MARs than in control lines without flanking MARs [1]. MARs have similar effects in plant cell systems. For example, in a tobacco cell culture system, we have shown that a heterologous MAR (a yeast MAR that binds weakly to the tobacco nuclear matrix) increases reporter gene expression by 12-fold, and a strong matrix-binding tobacco MAR increases reporter gene expression by 60-fold [3]. The mechanisms by which MARs increase reporter gene expression are unknown. MARs do not appear to act as typical enhancers, as they have little effect in transient expression assays [1, 2]. Most proposed mechanisms involve chromatin structure [1, 2]. According to one model, MARs affect transgene expression by creating independent, topologically isolated domains. The transgenes in these independent domains are insulated from chromatin structure of the native domains (either transcriptionally active or repressed) into which they become incorporated. If transgenic MARs do act by creating independent domains containing the transgene, the question remains of why the independent domains have a transcriptionally active chromatin structure. We have previously speculated that independent domains created by cloned MARs may have transcriptionally active chromatin structures because they are too small to form stable, transcriptionally repressed, condensed chromatin fibers in vivo [2, 3]. In our previous experiments, the putative domain formed by cloned MARs contains 3 kb of DNA, enough to form 16 plant nucleosomes. Even fewer nucleosomes may form on the transgenes in vivo, as close proximity to the nuclear matrix may sterically inhibit nucleosome formation. As the structure of the 30-nm chromatin fiber is not well understood, the number of nucleosomes necessary to form a stable, transcriptionally repressed structure is unknown. Formation of folded chromatin fibers presumably is dependent upon nucleosome–nucleosome interactions. If we consider the solenoid model of the 30-nm fiber (six nucleosomes per turn of the solenoid), it is logical to assume that a minimum of 12 nucleosomes would be required to form a stable, folded structure. This would allow each nucleosome to interact with at least one nucleosome other than its linear neighbors. Further turns of the solenoid would be expected to further stabilize the structure. For example, in a structure containing 18 nucleosomes (three turns of the solenoid), the nucleosomes of the middle turn could interact with nucleosomes of the outside two turns of the solenoid. Carruthers et al. [4] have provided evidence that a reconstituted linear DNA fragment containing 12 nucleosomes can form a stable, folded structure, but the relationship of this structure to the 30-nm fiber is unclear. The work of Butler and Thomas [5] indicates that more nucleosomes may be required. These workers observed a change in the hydrodynamic properties of native, rat liver nucleosome oligomers above the size of 50 nucleosomes. They attributed the change to higher-order folding. The considerations mentioned above suggest that in our previous work [2, 3] the 16 nucleosomes that would be expected to form on 3 kb of DNA bounded by MARs may be below or near the minimum required to form stable, folded chromatin fibers in vivo. Based on these ideas concerning the stability of higher-order structure in chromatin, we have asked if the enhancement of transgene expression by MARs can be counteracted by increasing the amount of DNA in the putative loop domain to a point at which a stable condensed chromatin fiber could be formed. In order to answer this question we have transformed plant cells in culture by microprojectile bombardment as we have previously described [3]. A co-transformation procedure is used in which a selectable marker (NPTII conferring kanamycin resistance) is carried on a separate plasmid from the reporter gene. As in our previous experiments we have used the reporter plasmids, pGHNC11 and pGHNC12 (Fig. 1A ). These plasmids contain the GUS reporter gene cassette flanked by the RB7-6 tobacco MAR and a control of the GUS cassette without flanking MARs. We also used a plasmid (pNMCS1 in Fig. 1A) in which λ DNA has been inserted between the GUS reporter cassette and the MAR. This ‘spacer’ DNA would increase the size of the putative MAR-bounded loop domain to 52 nucleosomes. The λ DNA (a 6.6-kb HindIII fragment, nucleotides 37586–44141) has an AT content of 51% and does not bind to the tobacco nuclear matrix (data not shown). A control plasmid (pNMCS2) with the λ DNA and the GUS reporter cassette but no MARs was also used. Kanamycin-resistant transformed cells were grown in liquid culture for 2 months with transfers every 7 days. At this time, cells were harvested and protein extracts were made in order to measure GUS specific activity [3]. Fig. 1B shows the results of the GUS specific activity measurements. Increasing the amount of DNA in the putative independent domain to a size that would support 8.6 turns of a nucleosome solenoid does not diminish the effect of MARs on enhancing reporter gene expression. The specific activity of the MAR-SPACER-GUS-MAR transformed cell lines is slightly higher than that in the MAR-GUS-MAR lines, but the difference is not statistically significant. In the lines transformed with constructs lacking MARs, the GUS activity was slightly lower in the SPACER-GUS lines than in the GUS-only lines, but again, this difference was not statistically significant. Because most DNA constructs used to test the effects of MARs on transgene expression contain amounts of DNA between the MARs that would be sufficient to form only a few nucleosomes [1-3], we hypothesized that the mechanism of MAR activity is the formation of loop domains that are too small to form a stable, transcriptionally repressed chromatin fiber. Increasing the amount of DNA in the putative MAR-bounded loop domains to a size that would accommodate 52 nucleosomes (nearly nine turns of a nucleosome solenoid) does not diminish the MAR-mediated enhancement of transgene expression in tobacco cells in culture. In vitro data indicate that 52 nucleosomes would be more than enough to support a stable, condensed chromatin fiber [5]. Thus, the explanation for MAR enhancement of transgene expression must lie elsewhere. We also note that the 5′ MAR is able to influence transgene expression at a fairly great distance (at least 6.6 kb from the promoter). It could be argued that the MAR located 3′ to the transgene is by itself causing the enhancement of transgene expression. But we have previously shown [2, 3] that both 5′ and 3′ flanking MARs are necessary to get the full MAR effect on increasing transgene expression. This work was supported by grant 9418491 from the USA National Science Foundation and by the North Carolina State University Agricultural Research Service. DA - 2001/5/4/ PY - 2001/5/4/ DO - 10.1016/s0014-5793(01)02406-1 SP - 66-67 ER - TY - PAT TI - Acid-inducible promoters for gene expression AU - Kullen, M. J. AU - Klaenhammer, T. R. C2 - 2001/// DA - 2001/// PY - 2001/// ER - TY - JOUR TI - Quantitative trait locus mapping of fitness-related traits in Drosophila melanogaster AU - Wayne, ML AU - Hackett, JB AU - Dilda, CL AU - Nuzhdin, SV AU - Pasyukova, EG AU - MacKay, TFC T2 - GENETICAL RESEARCH AB - We examined the genetic architecture of four fitness-related traits (reproductive success, ovariole number, body size and early fecundity) in a panel of 98 Oregon-R x 2b3 recombinant inbred lines (RILs). Highly significant genetic variation was observed in this population for female, but not male, reproductive success. The cross-sex genetic correlation for reproductive success was 0.20, which is not significantly different from zero. There was significant genetic variation segregating in this cross for ovariole number, but not for body size or early fecundity. The RILs were genotyped for cytological insertion sites of roo transposable elements, yielding 76 informative markers with an average spacing of 3.2 cM. Quantitative trait loci (QTL) affecting female reproductive success and ovariole number were mapped using a composite interval mapping procedure. QTL for female reproductive success were located at the tip of the X chromosome between markers at cytological locations 1B and 3E; and on the left arm of chromosome 2 in the 30D-38A cytological region. Ovariole number QTL mapped to cytological intervals 62D-69D and 98A-98E, both on the third chromosome. The regions harbouring QTL for female reproductive success and ovariole number were also identified as QTL for longevity in previous studies with these lines. DA - 2001/2// PY - 2001/2// DO - 10.1017/S0016672300004894 VL - 77 IS - 1 SP - 107-116 SN - 0016-6723 ER - TY - JOUR TI - Performance of a divergence time estimation method under a probabilistic model of rate evolution AU - Kishino, H AU - Thorne, JL AU - Bruno, WJ T2 - MOLECULAR BIOLOGY AND EVOLUTION AB - Rates of molecular evolution vary over time and, hence, among lineages. In contrast, widely used methods for estimating divergence times from molecular sequence data assume constancy of rates. Therefore, methods for estimation of divergence times that incorporate rate variation are attractive. Improvements on a previously proposed Bayesian technique for divergence time estimation are described. New parameterization more effectively captures the phylogenetic structure of rate evolution on a tree. Fossil information and other evidence can now be included in Bayesian analyses in the form of constraints on divergence times. Simulation results demonstrate that the accuracy of divergence time estimation is substantially enhanced when constraints are included. DA - 2001/3// PY - 2001/3// DO - 10.1093/oxfordjournals.molbev.a003811 VL - 18 IS - 3 SP - 352-361 SN - 0737-4038 KW - molecular clock KW - phylogeny KW - Markov chain Monte Carlo KW - Metropolis-Hastings algorithm ER - TY - JOUR TI - Functional genomics of odor-guided behavior in Drosophila melanogaster AU - Anholt, RRH AU - Fanara, JJ AU - Fedorowicz, GM AU - Ganguly, I AU - Kulkarni, NH AU - Mackay, TFC AU - Rollmann, SM T2 - CHEMICAL SENSES AB - The avoidance response to repellent odorants in Drosophila melanogaster, a response essential for survival, provides an advantageous model for studies on the genetic architecture of olfactory behavior. Transposon tagging in a highly inbred strain of flies in combination with a rapid and simple statistical behavioral assay enables the identification of not only large phenotypic effects, but also small aberrations from wild-type avoidance behavior. The recent completion of the sequence of the Drosophila genome facilitates the molecular characterization of transposon-tagged genes and correlation between gene expression and behavior in smell-impaired (smi) mutant lines. Quantitative genetic analyses of a collection of smi lines in a co-isogenic background revealed an extensive network of epistatic interactions among genes that shape the olfactory avoidance response. Candidate genes for several of these transposon-tagged smi loci implicate genes that mediate odorant recognition, including a novel odorant binding protein; signal propagation, including a voltage-gated sodium channel; and a protein containing multiple leucine rich repeats and PDZ domains likely to be involved in postsynaptic organization in the olfactory pathway. Several novel genes of unknown function have also been implicated, including a novel tyrosine-regulated protein kinase. The discovery and characterization of novel gene products that have major, hitherto unappreciated effects on olfactory behavior will provide new insights in the generation and regulation of odor-guided behavior. The identification and functional characterization of proteins encoded by smi genes that form part of the olfactory subgenome and correlation of polymorphisms in these genes with variation in odor-guided behavior in natural populations will advance our understanding of the genetic architecture of chemosensory behavior. DA - 2001/2// PY - 2001/2// DO - 10.1093/chemse/26.2.215 VL - 26 IS - 2 SP - 215-221 SN - 0379-864X ER - TY - JOUR TI - The scientific basis of Lactobacillus acidophilus NCFM functionality as a probiotic AU - Sanders, ME AU - Klaenhammer, TR T2 - JOURNAL OF DAIRY SCIENCE AB - Lactobacillus acidophilus NCFM is a probiotic strain available in conventional foods (milk, yogurt, and toddler formula) and dietary supplements. Its commercial availability in the United States since the mid-1970s is predicated on its safety, its amenability to commercial manipulation, and its biochemical and physiological attributes presumed to be important to human probiotic functionality. The strain has been characterized in vitro, in animal studies, and in humans. NCFM is the progenitor of the strain being used for complete chromosome sequencing and therefore will be a cornerstone strain for understanding the relationship between genetics and probiotic functionality. Both phenotypic and genotypic techniques have verified its taxonomic status as a type A1 L. acidophilus strain. It adheres to Caco-2 and mucus-secreting HT-29 cell culture systems, produces antimicrobial compounds, and is amenable to genetic manipulation and directed DNA introduction. NCFM survives gastrointestinal tract transit in both healthy and diseased populations. NCFM inhibits aberrant crypt formation in mutagenized rats, indicative of activity that could decrease the risk of colon cancer. A blend of probiotic strains containing NCFM decreased the incidence of pediatric diarrhea. NCFM led to a significant decrease in levels of toxic amines in the blood of dialysis patients with small bowel bacterial overgrowth. At adequate daily feeding levels, NCFM may facilitate lactose digestion in lactose-intolerant subjects. Further validation of the probiotic properties of NCFM in humans and clarification of its mechanisms of probiotic action are needed to better understand the role this strain might play in promoting human health. DA - 2001/2// PY - 2001/2// DO - 10.3168/jds.S0022-0302(01)74481-5 VL - 84 IS - 2 SP - 319-331 SN - 0022-0302 KW - Lactobacillus acidophilus NCFM KW - probiotic KW - functional food ER - TY - JOUR TI - The genetic architecture of odor-guided behavior in Drosophila melanogaster AU - Anholt, RRH AU - Mackay, TFC T2 - BEHAVIOR GENETICS DA - 2001/1// PY - 2001/1// DO - 10.1023/A:1010201723966 VL - 31 IS - 1 SP - 17-27 SN - 0001-8244 KW - olfaction KW - chemoreception KW - quantitative genetics KW - P-element insertional mutagenesis KW - epistasis KW - functional genomics ER - TY - JOUR TI - Structure of linkage disequilibrium and phenotypic associations in the maize genome AU - Remington, DL AU - Thornsberry, JM AU - Matsuoka, Y AU - Wilson, LM AU - Whitt, , SR AU - Doeblay, J AU - Kresovich, S AU - Goodman, MM AU - Buckler, ES T2 - PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA AB - Association studies based on linkage disequilibrium (LD) can provide high resolution for identifying genes that may contribute to phenotypic variation. We report patterns of local and genome-wide LD in 102 maize inbred lines representing much of the worldwide genetic diversity used in maize breeding, and address its implications for association studies in maize. In a survey of six genes, we found that intragenic LD generally declined rapidly with distance ( r 2 < 0.1 within 1500 bp), but rates of decline were highly variable among genes. This rapid decline probably reflects large effective population sizes in maize during its evolution and high levels of recombination within genes. A set of 47 simple sequence repeat (SSR) loci showed stronger evidence of genome-wide LD than did single-nucleotide polymorphisms (SNPs) in candidate genes. LD was greatly reduced but not eliminated by grouping lines into three empirically determined subpopulations. SSR data also supplied evidence that divergent artificial selection on flowering time may have played a role in generating population structure. Provided the effects of population structure are effectively controlled, this research suggests that association studies show great promise for identifying the genetic basis of important traits in maize with very high resolution. DA - 2001/9/25/ PY - 2001/9/25/ DO - 10.1073/pnas.201394398 VL - 98 IS - 20 SP - 11479-11484 SN - 0027-8424 ER - TY - JOUR TI - Structure and characterization of the crown gall opines heliopine, vitopine and rideopine AU - Chilton, WS AU - Petit, A AU - Chilton, MD AU - Dessaux, Y T2 - PHYTOCHEMISTRY AB - The crown gall opines heliopine from tumors induced by octopine type Agrobacterium tumefaciens strains A6, A136(pTiB6-806), E9, A652 and 1590-1 and vitopine from tumor induced by grapevine strains S4 and T2 are identical to synthetic N 2-(1′R-carboxyethyl)-l-glutamine. Tumors produced by strains S4 and T2 do not contain octopine or lysopine, but they do contain heliopine and the new opine ridéopine identified as N-(4′-aminobutyl)-d-glutamic acid. Grapevine strains S4 and T2 grow normally on tumor heliopine or synthetic heliopine and on tumor and synthetic ridéopine as well as on ridéopine lactam as sole carbon source. While octopine strains A6 and A136(pTiB6-806) do not grow on heliopine, mutant colonies do appear after a few weeks. Heliopine catabolism by octopine strains is not induced by octopine. DA - 2001/9// PY - 2001/9// DO - 10.1016/S0031-9422(01)00166-2 VL - 58 IS - 1 SP - 137-142 SN - 0031-9422 KW - crown gall KW - Agrobacterium tumefaciens KW - grapevine strains KW - opines KW - rideopine KW - N-(4 '-aminobutyl)-D-glutamic acid KW - heliopine KW - vitopine KW - N-2-(1 ' R-carboxyethyl)-L-glutamine ER - TY - JOUR TI - Silencing of a meristematic gene using geminivirus-derived vectors AU - Peele, C AU - Jordan, CV AU - Muangsan, N AU - Turnage, M AU - Egelkrout, E AU - Eagle, P AU - Hanley-Bowdoin, L AU - Robertson, D T2 - PLANT JOURNAL AB - Geminiviruses are DNA viruses that replicate and transcribe their genes in plant nuclei. They are ideal vectors for understanding plant gene function because of their ability to cause systemic silencing in new growth and ease of inoculation. We previously demonstrated DNA episome-mediated gene silencing from a bipartite geminivirus in Nicotiana benthamiana. Using an improved vector, we now show that extensive silencing of endogenous genes can be obtained using less than 100 bp of homologous sequence. Concomitant symptom development varied depending upon the target gene and insert size, with larger inserts producing milder symptoms. In situ hybridization of silenced tissue in attenuated infections demonstrated that silencing occurs in cells that lack detectable levels of viral DNA. A mutation confining the virus to vascular tissue produced extensive silencing in mesophyll tissue, further demonstrating that endogenous gene silencing can be separated from viral infection. We also show that two essential genes encoding a subunit of magnesium chelatase and proliferating cell nuclear antigen (PCNA) can be silenced simultaneously from different components of the same viral vector. Immunolocalization of silenced tissue showed that the PCNA protein was down-regulated throughout meristematic tissues. Our results demonstrate that geminivirus-derived vectors can be used to study genes involved in meristem function in intact plants. DA - 2001/8// PY - 2001/8// DO - 10.1046/j.1365-313x.2001.01080.x VL - 27 IS - 4 SP - 357-366 SN - 0960-7412 KW - Geminiviruses KW - homology-dependent gene silencing KW - meristems KW - functional genomics KW - plant DNA viruses KW - magnesium chelatase KW - proliferating cell nuclear antigen ER - TY - JOUR TI - Overlapping plant signal transduction pathways induced by a parasitic nematode and a rhizobial endosymbiont AU - Koltai, H AU - Dhandaydham, M AU - Opperman, C AU - Thomas, J AU - Bird, D T2 - MOLECULAR PLANT-MICROBE INTERACTIONS AB - Root-knot nematodes and rhizobia establish interactions with roots characterized by the de novo induction of host structures, termed giant cells and nodules, respectively. Two transcription regulators, PHAN and KNOX, required for the establishment of meristems were previously shown to be expressed in tomato giant cells. We isolated the orthologues of PHAN and KNOX (Mt-phan and Mt-knox-1) from the model legume Medicago truncatula, and established the spatial distribution of their expression in situ. We confirmed that Mt-phan and Mt-knox-1 are expressed in lateral root initials and in nematode-induced giant cells and showed that they are expressed in nodules induced by Sinorhizobium meliloti. Expression of both genes becomes spatially restricted as the nodules develop. We further examined nematode feeding sites for the expression of two genes involved in nodule formation, ccs52 (encodes a mitotic inhibitor) and ENOD40 (encodes an early, nodulation mitogen), and found transcripts of both genes to be present in and around giant cells induced in Medicago. Collectively, these results reveal common elements of host responses to mutualistic and parasitic plant endosymbionts and imply that overlapping regulatory pathways lead to giant cells and nodules. We discuss these pathways in the context of phytohormones and parallels between beneficial symbiosis and disease. DA - 2001/10// PY - 2001/10// DO - 10.1094/MPMI.2001.14.10.1168 VL - 14 IS - 10 SP - 1168-1177 SN - 0894-0282 KW - cytokinin KW - Meloidogyne incognita KW - Nod factor KW - PHANTASTICA KW - polar auxin flow KW - rough sheath2 ER - TY - JOUR TI - Map-based cloning of quantitative trait loci: progress and prospects AU - Remington, D. L. AU - Ungerer, M. C. AU - Purugganan, M. D. T2 - Genetical Research DA - 2001/// PY - 2001/// VL - 78 IS - 3 SP - 213-218 ER - TY - JOUR TI - Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination AU - Russell, WM AU - Klaenhammer, TR T2 - APPLIED AND ENVIRONMENTAL MICROBIOLOGY AB - ABSTRACT An efficient method is described for the generation of site-specific chromosomal integrations in Lactobacillus acidophilus and Lactobacillus gasseri . The strategy is an adaptation of the lactococcal pORI system (K. Leenhouts, G. Venema, and J. Kok, Methods Cell Sci. 20:35–50, 1998) and relies on the simultaneous use of two plasmids. The functionality of the integration strategy was demonstated by the insertional inactivation of the Lactobacillus acidophilus NCFM lacL gene encoding β-galactosidase and of the Lactobacillus gasseri ADH gusA gene encoding β-glucuronidase. DA - 2001/9// PY - 2001/9// DO - 10.1128/AEM.67.9.4361-4364.2001 VL - 67 IS - 9 SP - 4361-4364 SN - 0099-2240 ER - TY - JOUR TI - Analysis of the genetic switch and replication region of a P335-type bacteriophage with an obligate lytic lifestyle on Lactococcus lactis AU - Madsen, SM AU - Mills, D AU - Djordjevic, G AU - Israelsen, H AU - Klaenhammer, TR T2 - APPLIED AND ENVIRONMENTAL MICROBIOLOGY AB - The DNA sequence of the replication module, part of the lysis module, and remnants of a lysogenic module from the lytic P335 species lactococcal bacteriophage phi31 was determined, and its regulatory elements were investigated. The identification of a characteristic genetic switch including two divergent promoters and two cognate repressor genes strongly indicates that phi31 was derived from a temperate bacteriophage. Regulation of the two early promoters was analyzed by primer extension and transcriptional promoter fusions to a lacLM reporter. The regulatory behavior of the promoter region differed significantly from the genetic responses of temperate Lactococcus lactis phages. The cro gene homologue regulates its own production and is an efficient repressor of cI gene expression. No detectable cI gene expression could be measured in the presence of cro. cI gene expression in the absence of cro exerted minor influences on the regulation of the two promoters within the genetic switch. Homology comparisons revealed a replication module which is most likely expressed from the promoter located upstream of the cro gene homologue. The replication module encoded genes with strong homology to helicases and primases found in several Streptococcus thermophilus phages. Downstream of the primase homologue, an AT-rich noncoding origin region was identified. The characteristics and location of this region and its ability to reduce the efficiency of plaquing of phi31 10(6)-fold when present at high copy number in trans provide evidence for identification of the phage origin of replication. Phage phi31 is an obligately lytic phage that was isolated from commercial dairy fermentation environments. Neither a phage attachment site nor an integrase gene, required to establish lysogeny, was identified, explaining its lytic lifestyle and suggesting its origin from a temperate phage ancestor. Several regions showing extensive DNA and protein homologies to different temperate phages of Lactococcus, Lactobacillus, and Streptococcus were also discovered, indicating the likely exchange of DNA cassettes through horizontal gene transfer in the dynamic ecological environment of dairy fermentations. DA - 2001/3// PY - 2001/3// DO - 10.1128/AEM.67.3.1128-1139.2001 VL - 67 IS - 3 SP - 1128-1139 SN - 1098-5336 ER - TY - JOUR TI - An analysis of polygenes affecting wing shape on chromosome 2 in Drosophila melanogaster AU - Weber, K. AU - Eisman, R. AU - Higgins, S. AU - Morey, L. AU - Patty, A. AU - Tausek, M. AU - Zeng, Z. B. T2 - Genetics DA - 2001/// PY - 2001/// VL - 159 IS - 3 SP - 1045-1057 ER - TY - JOUR TI - A general polyploid model for analyzing gene segregation in outcrossing tetraploid species AU - Wu, R. L. AU - Gallo-Meagher, M. AU - Littell, R. C. AU - Zeng, Z. B. T2 - Genetics DA - 2001/// PY - 2001/// VL - 159 IS - 2 SP - 869-882 ER - TY - JOUR TI - The suitability of calcein to mark poeciliid fish and a new method of detection AU - Leips, J AU - Baril, CT AU - Rodd, FH AU - Reznick, DN AU - Bashey, F AU - Visser, GJ AU - Travis, J T2 - TRANSACTIONS OF THE AMERICAN FISHERIES SOCIETY AB - The suitability of calcein as a marker for population studies depends on (1) the assumption that marked individuals have unaltered viability, (2) the fidelity of the calcein label, and (3) the facility with which calcein can be used. We examined the effects of calcein on survival, growth, and the timing and size at sexual maturity of least killifish Heterandria formosa and present a new method for detecting calcein. To test the assumption that marked individuals have unaltered viability, juvenile least killifish were immersed for 24 h in a 250-mg/L solution of calcein. A control group of same-aged juveniles was immersed in the same volume of water for 24 h without calcein. All individuals were then removed and reared individually in separate containers. Upon examination under an epifluorescent microscope, all calcein-treated fish showed fluorescent green marks on their scales and fin rays, whereas controls did not. The calcein treatment had no significant effect on growth and survival through 9 weeks nor on the age and size at maturity. We also designed a portable detector (uses InGaN blue LEDs as a light source) for distinguishing calcein-marked individuals; using either this new detector or a standard epifluorescent microscope, the fluorescent mark was visible on the test fish for up to 5 weeks in the laboratory, although some individuals required remarking (due to fading) at 2–3 weeks postimmersion. The calcein tag was also visible in the vertebrae of ethanol-preserved specimens for up to 6 years, provided specimens were stored in the dark. DA - 2001/5// PY - 2001/5// DO - 10.1577/1548-8659(2001)130<0501:TSOCTM>2.0.CO;2 VL - 130 IS - 3 SP - 501-507 SN - 0002-8487 ER - TY - JOUR TI - High-throughput AFLP analysis using infrared dye-labeled primers and an automated DNA sequencer AU - Myburg, AA AU - Remington, DL AU - DM O'Malley, AU - Sederoff, RR AU - Whetten, RW T2 - BIOTECHNIQUES AB - Amplified fragment length polymorphism (AFLP) analysis is currently the most powerful and efficient technique for the generation of large numbers of anonymous DNA markers in plant and animal genomes. We have developed a protocol for high-throughput AFLP analysis that allows up to 70 000 polymorphic marker genotype determinations per week on a single automated DNA sequencer. This throughput is based on multiplexed PCR amplification of AFLP fragments using two different infrared dyelabeled primer combinations. The multiplexed AFLPs are resolved on a two-dye, model 4200 LI-COR ® automated DNA sequencer, and the digital images are scored using semi-automated scoring software specifically designed for complex AFLP banding patterns (AFLP-Quantar™). Throughput is enhanced by using high-quality genomic DNA templates obtained by a 96-well DNA isolation procedure. DA - 2001/2// PY - 2001/2// DO - 10.2144/01302tt04 VL - 30 IS - 2 SP - 348-+ SN - 0736-6205 ER - TY - JOUR TI - Genetic evidence and the origin of maize (Biology, archaeology) AU - Bennetzen, J AU - Buckler, E AU - Chandler, V AU - Doebley, J AU - Dorweiler, J AU - Gaut, B AU - Freeling, M AU - Hake, S AU - Kellogg, E AU - Poethig, RS AU - Walbot, V AU - Wessler, S T2 - LATIN AMERICAN ANTIQUITY AB - The origin of maize has been a topic of interest to both biologists and archaeologists. During the twentieth century, the view point that maize is a domesticated form of teosinte received convincing support from biological data and is now broadly accepted among biologists familiar with the issues and data. There is no support of any kind for an alternative view that maize is a hybrid of the grasses Zea diploperennis and Tripsacum . DA - 2001/3// PY - 2001/3// DO - 10.2307/971759 VL - 12 IS - 1 SP - 84-86 SN - 2325-5080 ER - TY - JOUR TI - Evolution: A complement for evolutionary genetics AU - Gibson, G AU - Palsson, A T2 - CURRENT BIOLOGY AB - Developmental geneticists' contribution to the study of the evolution of morphological divergence has proceeded along two lines: comparative analysis of gene expression and quantitative genetics. Recent studies highlight how complementation tests between species can bridge the gap between these approaches. DA - 2001/1/23/ PY - 2001/1/23/ DO - 10.1016/S0960-9822(01)00014-8 VL - 11 IS - 2 SP - R74-R76 SN - 0960-9822 ER - TY - JOUR TI - Evolution of proteasomal ATPases AU - Wollenberg, K AU - Swaffield, JC T2 - MOLECULAR BIOLOGY AND EVOLUTION AB - In eukaryotic cells, the majority of proteins are degraded via the ATP-dependent ubiquitin/26S proteasome pathway. The proteasome is the proteolytic component of the pathway. It is a very large complex with a mass of around 2.5 MDa, consisting of at least 62 proteins encoded by 31 genes. The eukaryotic proteasome has evolved from a simpler archaebacterial form, similar in structure but containing only three different peptides. One of these peptides is an ATPase belonging to the AAA (Triple-A) family of ATPASES: Gene duplication and diversification has resulted in six paralogous ATPases being present in the eukaryotic proteasome. While sequence analysis studies clearly show that the six eukaryotic proteasomal ATPases have evolved from the single archaebacterial proteasomal ATPase, the deep node structures of the phylogenetic constructions lack resolution. Incorporating physical data to provide support for alternative phylogenetic hypotheses, we have constructed a model of a possible evolutionary history of the proteasomal ATPASES: DA - 2001/6// PY - 2001/6// DO - 10.1093/oxfordjournals.molbev.a003897 VL - 18 IS - 6 SP - 962-974 SN - 1537-1719 KW - proteasome KW - proteasomal ATPases KW - multiprotein complex KW - cross-linking KW - AAA ATPase KW - evolution ER - TY - JOUR TI - An allele-specific hammerhead ribozyme gene therapy for a porcine model of autosomal dominant retinitis pigmentosa AU - Shaw, L. C. AU - Skold, A. AU - Wong, F. AU - Petters, R. AU - Hauswirth, W. AU - Lewin, A. S. T2 - Molecular Vision DA - 2001/// PY - 2001/// VL - 7 IS - 2 SP - 6-13 ER - TY - JOUR TI - A screen for sequences up-regulated during heterocyst development in Anabaena sp strain PCC 7120 AU - Curtis, SE AU - Hebbar, PB T2 - ARCHIVES OF MICROBIOLOGY DA - 2001/5// PY - 2001/5// DO - 10.1007/s002030100267 VL - 175 IS - 5 SP - 313-322 SN - 0302-8933 KW - Anabaena sp strain PCC 7120 KW - cyanobacteria KW - heterocyst development KW - nitrogen starvation KW - genome screening KW - up-regulated genes ER - TY - JOUR TI - The 5 ' end of the pea ferredoxin-1 mRNA mediates rapid and reversible light-directed changes in translation in tobacco AU - Hansen, ER AU - Petracek, ME AU - Dickey, LF AU - Thompson, WF T2 - PLANT PHYSIOLOGY AB - Abstract Ferredoxin-1 (Fed-1) mRNA contains an internal light response element (iLRE) that destabilizes mRNA when light-grown plants are placed in darkness. mRNAs containing this element dissociate from polyribosomes in the leaves of transgenic tobacco (Nicotiana tabacum) plants transferred to the dark for 2 d. Here, we report in vivo labeling experiments with a chloramphenicol acetyl transferase mRNA fused to theFed-1 iLRE. Our data indicate that theFed-1 iLRE mediates a rapid decline in translational efficiency and that iLRE-containing mRNAs dissociate from polyribosomes within 20 min after plants are transferred to darkness. Both events occur before the decline in mRNA abundance, and polyribosome association is rapidly reversible if plants are re-illuminated. These observations support a model in which Fed-1 mRNA in illuminated leaves is stabilized by its association with polyribosomes, and/or by translation. In darkness a large portion of the mRNA dissociates from polyribosomes and is subsequently degraded. We also show that a significant portion of total tobacco leaf mRNA is shifted from polyribosomal to non-polyribosomal fractions after 20 min in the dark, indicating that translation of other mRNAs is also rapidly down-regulated in response to darkness. This class includes some, but not all, cytoplasmic mRNAs encoding proteins involved in photosynthesis. DA - 2001/2// PY - 2001/2// DO - 10.1104/pp.125.2.770 VL - 125 IS - 2 SP - 770-778 SN - 0032-0889 ER - TY - JOUR TI - Vertebrate serpins: Construction of a conflict-free phylogeny by combining exon-intron and diagnostic site analyses AU - Ragg, H AU - Lokot, T AU - Kamp, PB AU - Atchley, WR AU - Dress, A T2 - MOLECULAR BIOLOGY AND EVOLUTION AB - A combination of three independent biological features, genomic organization, diagnostic amino acid sites, and rare indels, was used to elucidate the phylogeny of the vertebrate serpin (serine protease inhibitor) superfamily. A strong correlation between serpin gene families displaying (1) a conserved exon-intron pattern and (2) family-specific combinations of amino acid residues at specific sites suggests that present-day vertebrates encompass six serpin gene families which evolved from primordial genes by massive intron insertion before or during early vertebrate radiation. Introns placed at homologous positions in the gene sequences in combination with diagnostic sequence characters may also constitute a reliable kinship indicator for other protein superfamilies. DA - 2001/4// PY - 2001/4// DO - 10.1093/oxfordjournals.molbev.a003838 VL - 18 IS - 4 SP - 577-584 SN - 1537-1719 KW - molecular evolution KW - serpins KW - exon-intron structure KW - diagnostic sites KW - heparin cofactor II ER - TY - JOUR TI - The role of cell differentiation state and HMG-I/Y in the expression of transgenes flanked by matrix attachment regions AU - Ascenzi, R AU - Ingram, JL AU - Massel, M AU - Thompson, WF AU - Spiker, S AU - Weissinger, AK T2 - TRANSGENIC RESEARCH DA - 2001/// PY - 2001/// DO - 10.1023/A:1012082602587 VL - 10 IS - 5 SP - 465-470 SN - 0962-8819 KW - cell differentiation and proliferation KW - chromatin KW - matrix KW - scaffold attachment region (MAR/SAR) KW - plant high mobility group-I/Y protein (HMG-I/Y) KW - transgene expression KW - tobacco ER - TY - JOUR TI - Leaky Lactococcus cultures that externalize enzymes and antigens independently of culture lysis and secretion and export pathways AU - Walker, SA AU - Klaenhammer, TR T2 - APPLIED AND ENVIRONMENTAL MICROBIOLOGY AB - ABSTRACT A novel system that leaks β-galactosidase (β-gal) without a requirement for secretion or export signals was developed in Lactococcus lactis by controlled expression of integrated phage holin and lysin cassettes. The late promoter of the lytic lactococcal bacteriophage φ31 is an 888-bp fragment (P 15A10 ) encoding the transcriptional activator. When a high-copy-number P 15A10 :: lacZ.st fusion was introduced into L. lactis strains C10, ML8, NCK203, and R1/r1t, high levels of the resultant β-gal activity were detected in the supernatant (approximately 85% of the total β-gal activity for C10, ML8, and NCK203 and 45% for R1/r1t). Studies showed that the phenotype resulted from expression of Tac31A from the P 15A10 fragment, which activated a homologous late promoter in prophages harbored by the lactococcal strains. Despite the high levels of β-gal obtained in the supernatant, the growth of the strains was not significantly affected, nor was there any evidence of severe membrane damage as determined by using propidium iodide or transmission electron microscopy. Integration of the holin-lysin cassette of phage r1t, under the control of the phage φ31 late promoter, into the host genome of MG1363 yielded a similar “leaky” phenotype, indicating that holin and lysin might play a critical role in the release of β-gal into the medium. In addition to β-gal, tetanus toxin fragment C was successfully delivered into the growth medium by this system. Interestingly, the X-prolyl dipeptidyl aminopeptidase PepXP (a dimer with a molecular mass of 176 kDa) was not delivered at significant levels outside the cell. These findings point toward the development of bacterial strains able to efficiently release relevant proteins and enzymes outside the cell in the absence of known secretion and export signals. DA - 2001/1// PY - 2001/1// DO - 10.1128/AEM.67.1.251-259.2001 VL - 67 IS - 1 SP - 251-259 SN - 1098-5336 ER - TY - JOUR TI - Generation of Intron-Containing, ER-Localized, Soluble-Modified Green Fluorescent Protein Genes for use in Plant Transformation AU - Mankin, S.L. AU - Thompson, W.F. T2 - Plant Molecular Biology Reporter DA - 2001/// PY - 2001/// DO - 10.1007/bf02824074 VL - 19 IS - 1 SP - 13–26 ER - TY - JOUR TI - Microsatellite variation in cassava (Manihot esculenta, Euphorbiaceae) and its wild relatives: Further evidence for a southern Amazonian origin of domestication AU - Olsen, KM AU - Schaal, BA T2 - AMERICAN JOURNAL OF BOTANY AB - Genetic variation at five microsatellite loci was used to investigate the evolutionary and geographical origins of cassava ( Manihot esculenta subsp. esculenta ) and the population structure of cassava's wild relatives. Two hundred and twelve individuals were sampled, representing 20 crop accessions, 27 populations of cassava's closest wild relative ( M. esculenta subsp. flabellifolia ), and six populations of a potentially hybridizing species ( M. pruinosa ). Seventy‐three alleles were observed across all loci and populations. These data indicate the following on cassava's origin: (1) genetic variation in the crop is a subset of that found in the wild M. esculenta subspecies, suggesting that cassava is derived solely from its conspecific wild relative. (2) Phenetic analyses group cassava with wild populations from the southern border of the Amazon basin, indicating this region as the likely site of domestication. (3) Manihot pruinosa , while closely related to M. esculenta (and possibly hybridizing with it where sympatric), is probably not a progenitor of the crop. Genetic differentiation among the wild populations is moderately high ( F ST = 0.42, ρ ST = 0.54). This differentiation has probably arisen primarily through random genetic drift (rather than mutation) following recent population divergence. DA - 2001/1// PY - 2001/1// DO - 10.2307/2657133 VL - 88 IS - 1 SP - 131-142 SN - 0002-9122 KW - cassava KW - Manihot esculenta KW - Manihot pruinosa KW - microsatellites KW - origin of domestication KW - population structure KW - wild relatives ER - TY - JOUR TI - The drosophila genes disconnected and disco-related are redundant with respect to larval head development and accumulation of mRNAs from deformed target genes AU - Mahaffey, J. W. AU - Griswold, C. M. AU - Cao, Q. M. T2 - Genetics DA - 2001/// PY - 2001/// VL - 157 IS - 1 SP - 225-236 ER - TY - JOUR TI - Quantitative trait loci for the monoamine-related traits heart rate and headless behavior in Drosophila melanogaster AU - Ashton, K. AU - Wagoner, A. P. AU - Carrillo, R. AU - Gibson, G. T2 - Genetics DA - 2001/// PY - 2001/// VL - 157 IS - 1 SP - 283-294 ER - TY - JOUR TI - Joint linkage and linkage disequilibrium mapping in natural populations AU - Wu, R. L. AU - Zeng, Z. B. T2 - Genetics DA - 2001/// PY - 2001/// VL - 157 IS - 2 SP - 899-909 ER - TY - JOUR TI - Dual interaction of a geminivirus replication accessory factor with a viral replication protein and a plant cell cycle regulator AU - Settlage, SB AU - Miller, AB AU - Gruissem, W AU - Hanley-Bowdoin, L T2 - VIROLOGY AB - Geminiviruses replicate their small, single-stranded DNA genomes through double-stranded DNA intermediates in plant nuclei using host replication machinery. Like most dicot-infecting geminiviruses, tomato golden mosaic virus encodes a protein, AL3 or C3, that greatly enhances viral DNA accumulation through an unknown mechanism. Earlier studies showed that AL3 forms oligomers and interacts with the viral replication initiator AL1. Experiments reported here established that AL3 also interacts with a plant homolog of the mammalian tumor suppressor protein, retinoblastoma (pRb). Analysis of truncated AL3 proteins indicated that pRb and AL1 bind to similar regions of AL3, whereas AL3 oligomerization is dependent on a different region of the protein. Analysis of truncated AL1 proteins located the AL3-binding domain between AL1 amino acids 101 and 180 to a region that also includes the AL1 oligomerization domain and the catalytic site for initiation of viral DNA replication. Interestingly, the AL3-binding domain was fully contiguous with the domain that mediates AL1/pRb interactions. The potential significance of AL3/pRb binding and the coincidence of the domains responsible for AL3, AL1, and pRb interactions are discussed. DA - 2001/1/20/ PY - 2001/1/20/ DO - 10.1006/viro.2000.0719 VL - 279 IS - 2 SP - 570-576 SN - 0042-6822 ER - TY - JOUR TI - Phylogenetic analyses of amino acid variation in the serpin proteins AU - Atchley, WR AU - Lokot, T AU - Wollenberg, K AU - Dress, A AU - Ragg, H T2 - MOLECULAR BIOLOGY AND EVOLUTION AB - Phylogenetic analyses of 110 serpin protein sequences revealed clades consistent with independent phylogenetic analyses based on exon-intron structure and diagnostic amino acid sites. Trees were estimated by maximum likelihood, neighbor joining, and partial split decomposition using both the BLOSUM 62 and Jones-Taylor-Thornton substitution matrices. Neighbor-joining trees gave results closest to those based on independent analyses using genomic and chromosomal data. The maximum-likelihood trees derived using the quartet puzzling algorithm were very conservative, producing many small clades that separated groups of proteins that other results suggest were related. Independent analyses based on exon-intron structure suggested that a neighbor-joining tree was more accurate than maximum-likelihood trees obtained using the quartet puzzling algorithm. DA - 2001/8// PY - 2001/8// DO - 10.1093/oxfordjournals.molbev.a003936 VL - 18 IS - 8 SP - 1502-1511 SN - 1537-1719 KW - serpins KW - molecular evolution KW - phylogeny KW - protein evolution KW - maximum likelihood KW - neighbor joining ER - TY - JOUR TI - Maize ribosome-inactivating protein inhibits normal development of Aspergillus nidulans and Aspergillus flavus AU - Nielsen, K AU - Payne, GA AU - Boston, RS T2 - MOLECULAR PLANT-MICROBE INTERACTIONS AB - The abundant maize kernel ribosome-inactivating protein 1 (RIP1) was tested for antifungal activity against Aspergillus nidulans and Aspergillus flavus. A microculture assay was developed to monitor fungal growth and development after treatment of conidia with RIP1 or control proteins. A striking decrease in hyphal proliferation was observed when conidia of A. nidulans, a genetically well-characterized nonpathogenic species, were treated with RIP1 protein. Treatment with a RIP1 mutant protein that lacked enzymatic ribosome-inactivating activity caused no observable effects. RIP1 treatment of conidia from the maize pathogen A. flavus resulted in increased hyphal branching. Examination of the branched hyphae after Congo red staining revealed only one growing hyphal tip per conidium. These results indicate that both fungi were affected by RIP1 treatment, but the lysis seen with treatment of A. nidulans was apparently avoided by A. flavus. A developmental time course revealed that both fungal species were affected by RIP1 at the postdivisional growth stage. The inhibitory activity of RIP1 against normal fungal growth is consistent with a biological function to protect the seed from fungal invasion. DA - 2001/2// PY - 2001/2// DO - 10.1094/mpmi.2001.14.2.164 VL - 14 IS - 2 SP - 164-172 SN - 0894-0282 KW - b-32 KW - plant defense KW - seed protein KW - Zea mays L ER - TY - JOUR TI - Differential antioxidant responses to norflurazon-induced oxidative stress in maize AU - Jung, S AU - Kernodle, SP AU - Scandalios, JG T2 - REDOX REPORT AB - This study examined the contribution of catalase (CAT) and superoxide dismutase (SOD) in the overall antioxidant response to norflurazon (NF)-induced oxidative stress in leaves, mesocotyls and scutella of maize (Zea mays). Maize catalase null mutants were used to provide insights into the role(s) of these isozymes. A substantial increase in Cat1 and Cat2 transcript levels occurred in NF-treated leaves in all maize lines examined. However, these two transcripts did not show a particular pattern of change in NF-treated scutella from 5-day postimbibition (dpi) and 18-day postpollination (dpp) maize. The NF-induced increase in Cat1 appeared to be dependent on excessive light energy caused by a lack of photoprotectant carotenoids. especially in leaves. In NF-treated leaves, the chloroplastic Cu/Zn-SOD-1 isozyme responded strongly compared to the cytosolic Cu/Zn-SOD and mitochondrial Mn-SOD-3 isozymes, suggesting the critical role of SOD-1 as a major component in chloroplastic antioxidant defenses. All SOD isozymes in the NF-treated scutella of various maize lines were consistent in their response to NF. The most significant increase was observed with Sod1 in NF-treated leaves; however, no significant Sod1 changes were observed in similarly treated scutella at 5 dpi and 18 dpp. These results suggest that the response of the Cat and Sod genes to NF is likely developmental and tissue-specific. DA - 2001/// PY - 2001/// DO - 10.1179/135100001101536454 VL - 6 IS - 5 SP - 311-317 SN - 1743-2928 ER - TY - JOUR TI - A multivalent pairing model of linkage analysis in autotetraploids AU - Wu, S. S. AU - Wu, R. L. AU - Ma, C. X. AU - Zeng, Z. B. AU - Yang, M. C. AU - Casella, G. T2 - Genetics DA - 2001/// PY - 2001/// VL - 159 IS - 3 SP - 1339-1350 ER - TY - JOUR TI - Quantitative trait loci in Drosophila AU - Mackay, TFC T2 - NATURE REVIEWS GENETICS DA - 2001/1// PY - 2001/1// DO - 10.1038/35047544 VL - 2 IS - 1 SP - 11-20 SN - 1471-0064 ER - TY - JOUR TI - Pulping and bleaching of CAD-deficient wood AU - Dimmel, DR AU - MacKay, JJ AU - Althen, EM AU - Parks, C AU - Sederoff, RR T2 - JOURNAL OF WOOD CHEMISTRY AND TECHNOLOGY AB - Mutant loblolly pine trees that are deficient in the enzyme cinnamyl alcohol dehydrogenase (CAD) have been obtained through directed breeding. The lignin in the wood of CAD-deficient trees has a different pool of precursors, resulting in high levels of pulping-resistant C-5 linkages. Wood from a 12-year-old CAD-deficient tree has been pulped under soda and kraft conditions in microdigestors. In comparison to a normal 12-year-old loblolly pine, the CAD-deficient wood was much more easily delignified. In addition, the pulp from CAD-deficient wood was as easy to bleach as a control pulp. The high reactivity of CAD-deficient wood may be related to the lignin size and phenolic content. The molecular weight of an isolated milled wood lignin from CAD-deficient pine was ∼35% less than that from a normal pine tree. DA - 2001/// PY - 2001/// DO - 10.1081/WCT-100102651 VL - 21 IS - 1 SP - 1-17 SN - 1532-2319 ER - TY - JOUR TI - Molecular diversity, structure and domestication of grasses AU - Buckler, E. S. AU - Thornsberry, J. M. AU - Kresovich, S. T2 - Genetical Research DA - 2001/// PY - 2001/// VL - 77 IS - 3 SP - 213-218 ER - TY - PAT TI - Matrix attachment regions AU - Michalowski, S. M. AU - Spiker, S. C2 - 2001/// DA - 2001/// PY - 2001/// ER - TY - JOUR TI - Identification and cloning of gusA, encoding a new beta-glucuronidase from Lactobacillus gasseri ADH AU - Russell, WM AU - Klaenhammer, TR T2 - APPLIED AND ENVIRONMENTAL MICROBIOLOGY AB - ABSTRACT The gusA gene, encoding a new β-glucuronidase enzyme, has been cloned from Lactobacillus gasseri ADH. This is the first report of a β-glucuronidase gene cloned from a bacterial source other than Escherichia coli . A plasmid library of L. gasseri chromosomal DNA was screened for complementation of an E. coli gus mutant. Two overlapping clones that restored β-glucuronidase activity in the mutant strain were sequenced and revealed three complete and two partial open reading frames. The largest open reading frame, spanning 1,797 bp, encodes a 597-amino-acid protein that shows 39% identity to β-glucuronidase (GusA) of E. coli K-12 (EC 3.2.1.31 ). The other two complete open reading frames, which are arranged to be separately transcribed, encode a putative bile salt hydrolase and a putative protein of unknown function with similarities to MerR-type regulatory proteins. Overexpression of GusA was achieved in a β-glucuronidase-negative L. gasseri strain by expressing the gusA gene, subcloned onto a low-copy-number shuttle vector, from the strong Lactobacillus P6 promoter. GusA was also expressed in E. coli from a pET expression system. Preliminary characterization of the GusA protein from crude cell extracts revealed that the enzyme was active across an acidic pH range and a broad temperature range. An analysis of other lactobacilli identified β-glucuronidase activity and gusA homologs in other L. gasseri isolates but not in other Lactobacillus species tested. DA - 2001/3// PY - 2001/3// DO - 10.1128/AEM.67.3.1253-1261.2001 VL - 67 IS - 3 SP - 1253-1261 SN - 0099-2240 ER - TY - JOUR TI - Expression of a chimeric retroviral-lipase mRNA confers enhanced lipolysis in a hibernating mammal AU - Bauer, V. W. AU - Squire, T. L. AU - Lowe, M. E. AU - Andrews, M. T. T2 - American Journal of Physiology DA - 2001/// PY - 2001/// VL - 281 IS - 4 SP - R1186-1192 ER - TY - JOUR TI - Developmental evolution: The unbearable likeness of beings AU - Gibson, G T2 - CURRENT BIOLOGY AB - Comparative studies have revealed a remarkable range of genetic changes in the mechanisms that pattern the nematode vulva. Two new studies identify genetic variation within nematode species that affects cell division and competence in vulval precursors. DA - 2001/5/1/ PY - 2001/5/1/ DO - 10.1016/S0960-9822(01)00190-7 VL - 11 IS - 9 SP - R345-R348 SN - 0960-9822 ER - TY - JOUR TI - Computing mating bull fertility from DHI nonreturn data AU - Clay, JS AU - McDaniel, BT T2 - JOURNAL OF DAIRY SCIENCE AB - Animal model methodology was used to compute yearly measures of relative fertility of Holstein AI mating bulls based upon 70-d nonreturn of first breedings as reported to U.S. DHIA from 1988 through 1997. Estimated Relative Conception Rates (ERCR) were computed for bulls with a minimum of 50 first breedings in a single year using variance ratios 45.5 for mating bull, 45.5 for animal genetic effects, and 31 for permanent environment. The model assumed repeatability across lactations of 0.05 and included fixed effects of herd-year-month bred and classes of parity, early lactation energy-corrected milk and days open when bred. Estimates of fertility were greater for breedings to cows that were young, had low early lactation production, and were in late stages of lactation. ERCR were expressed as difference in nonreturn from the average AI mating bull of herdmates. Values ranged from -18 to +13. For ERCR computed from a minimum of 1000 breedings, 90% were within four units of zero. Early ERCR computed from a few breedings in a single year were tested for ability to predict later ERCR computed from a minimum of 1000 different breedings. Early ERCR computed from 300 or more matings accurately predicted later independent ERCR. For yearly estimates each based upon a minimum of 1000 breedings, 8% changed more than three units, and 4% declined more than three units. Correlations between ERCR and predicted transmitting abilities protein and type production index were significant but accounted for little variance. Correlations between ERCR and other traits were not significant. DA - 2001/5// PY - 2001/5// DO - 10.3168/jds.S0022-0302(01)74585-7 VL - 84 IS - 5 SP - 1238-1245 SN - 0022-0302 KW - nonreturn KW - fertility KW - mating bull KW - animal model ER -