Gene-specific Analysis Research Articles

A note on joint versus gene-specific mixed model analysis of microarray gene expression data

Currently, linear mixed model analyses of expression microarray experiments are performed either in a gene-specific or global mode. The joint analysis provides more flexibility in terms of how parameters are fitted and estimated and tends to be more powerful than the gene-specific analysis. Here we show how to implement the gene-specific linear mixed model analysis as an exact algorithm for the joint linear mixed model analysis. The gene-specific algorithm is exact, when the mixed model equations can be partitioned into unrelated components: One for all global fixed and random effects and the others for the gene-specific fixed and random effects for each gene separately. This unrelatedness holds under three conditions: (1) any gene must have the same number of replicates or probes on all arrays, but these numbers can differ among genes; (2) the residual variance of the (transformed) expression data must be homogeneous or constant across genes (other variance components need not be homogeneous) and (3) the number of genes in the experiment is large. When these conditions are violated, the gene-specific algorithm is expected to be nearly exact.

Osteo-Promoter Database (OPD) – Promoter analysis in skeletal cells

BackgroundIncreasing our knowledge about the complex expression of genes in skeletal tissue will provide a better understanding of the physiology of skeletal cells. The study summarizes transcriptional regulation factors interacting and cooperating at promoter regions that regulate gene expression. Specifically, we analyzed A/T rich elements along the promoter sequences.DescriptionThe Osteo-Promoter Database (OPD) is a collection of genes and promoters expressed in skeletal cells. We have compiled a new viewer, OPD, as unique database developed and created as an accessible tool for skeletal promoter sequences. OPD can navigate to identify genes specific to skeletal cDNA databases and promoter analysis sites. OPD offers exclusive access to facilitate a dynamic extraction of promoters' gene-specific analyses in skeletal tissue. The data on promoters included in OPD contains cloned promoters or predicted promoters that were analyzed by bioinformatics tools. OPD offers MAR-analysis, which allocates A/T rich elements along these promoter sequences.ConclusionThe analysis leads to a better insight of proteins that bind to DNA, regulate DNA, and function in chromatin remodeling. The OPD is a distinctive tool for understanding the complex function of chromatin remodeling and transcriptional regulation of specific gene expression in skeletal tissue.

Increased GADD gene expression in human colon epithelial cells exposed to deoxycholate.

The colonic epithelium is often exposed to high concentrations of secondary bile acids, which stresses the epithelial cells, leading potentially to activation of stress-response genes. To examine this possibility in vitro, the purpose of this study was to determine if expression of certain growth arrest and DNA damage-inducible genes (GADD) is upregulated in human colonic epithelial cells exposed to deoxycholate (DOC). DNA macroarray screening of a small cluster of stress/apoptosis-related genes in DOC-treated HCT-116 colonocytes revealed clearly higher expression of only GADD45, which was confirmed by gene-specific relative RT-PCR analysis. Subsequently, it was found that DOC also increased GADD34 mRNA expression. However, mRNA expression of GADD153 was increased most markedly in DOC-treated HCT-116 colonocytes, which express wild-type p53. However, the upregulation of GADD34, GADD45, and GADD153 mRNA expression apparently did not require p53, based on the finding that DOC increased expression of all three GADD genes in HCT-15 colonocytes, which express mutant p53. In further studying GADD153 in particular, the effect of DOC on GADD153 mRNA was prevented by actinomycin-D (Act-D), but not by antioxidants or MAPK inhibitors. DOC also caused GADD153 protein to be expressed in close parallel with increased GADD153 mRNA expression. Induction of GADD153 protein by DOC was prevented by either anisomycin or cycloheximide. These findings suggest that DOC-induced upregulation of GADD153 mRNA expression occurred at the level of transcription without involving reactive oxygen species and MAPK signaling, and that the expression of GADD153 protein was due also to translation of pre-existing, and not just newly synthesized, mRNA.

Genome-wide Analysis of ARS (Autonomously Replicating Sequence) Binding Factor 1 (Abf1p)-mediated Transcriptional Regulation in Saccharomyces cerevisiae

Autonomously replicating sequence-binding factor-1 (Abf1p) is an essential sequence-specific transcription factor in Saccharomyces cerevisiae that participates in multiple nuclear events including DNA replication, transcription activation, and gene silencing. Numerous gene-specific analyses have implicated Abf1p in the transcriptional control of genes involved in a diverse range of cellular functions, leading to the notion that Abf1p acts as a global transcriptional regulator. Here we report findings from a genome-wide comparison of the gene expression profiles in the wild-type and abf1-1 temperature-sensitive mutant. The study identifies a total of 86 Abf1p-regulated genes (1.4% of the genome) of which 50 are activated and 36 are repressed by Abf1p. Interestingly, Abf1p binds to its own promoter in vivo and strongly represses its own transcription, suggesting a potential negative regulatory loop in Abf1p-mediated gene regulation. A comparison of our microarray data with the available databases reveals a significant overlap of genes regulated by Abf1p and those by several general transcription factors such as Mot1p and TAFs (TATA-binding protein-associated factors). Different mutant alleles of abf1 affect Abf1p-mediated transcription in a gene-dependent manner. Furthermore, Abf1p in vivo is associated with the promoter region of most Abf1p-activated but not with that of most Abf1p-repressed genes. Taken together, these results strongly suggest distinct underlying mechanisms by which Abf1p regulates gene expression.

Constrained Binding Site Diversity within Families of Transcription Factors Enhances Pattern Discovery Bioinformatics

Diverse computational and experimental efforts are required to elucidate the control circuitry regulating the transcription of human genes. The fusion of gene-specific promoter analyses with large microarray studies and bioinformatics advances has produced optimism that significant progress can be made in unravelling this complex network. Within bioinformatics, past emphasis for improved pattern discovery has been placed upon “phylogenetic footprinting”, the identification of sequences conserved over moderate periods of evolution (e.g. human and mouse comparisons). We introduce a new direction in bioinformatics based on the constraints imposed by the structures of DNA-binding proteins. For most structurally related families of transcription factors, there are clear similarities in the sequences of the sites to which they bind. On the basis of this observation, we construct familial binding profiles for well-characterized transcription factor families. The profiles are shown to classify correctly the structural class of mediating transcription factors for novel motifs in 88% of cases. By incorporating the familial profiles into pattern discovery procedures, we demonstrate that functional binding sites can be found in genomic sequences of dramatically greater length than is possible otherwise. Thus, incorporating familial models can overcome the signal-to-noise challenge that has hindered the transition from microarray data to regulatory control sequences for human genes. Biochemically motivated constraints upon sequence diversity of binding sites will complement the genetically motivated constraints imposed in “phylogenetic footprinting” algorithms.

Assessment of positional candidate genes myf5 and igf1 for growth on bovine chromosome 5 in commercial lines of Bos taurus.

Quantitative trait loci for growth traits in beef cattle have been previously reported and fine-mapped in three chromosomal regions of 0 to 30 cM, 55 to 70 cM, and 70 to 80 cM of bovine chromosome 5. In this study, we further examined the association between gene-specific single nucleotide polymorphisms (SNP) of two positional candidate genes, bovine myogenic factor 5 (myf5) and insulin-like growth factor-1 (igf1), in the QTL regions and the birth weight (BWT), preweaning average daily gain (PWADG), and average daily gain on feed (ADGF) in commercial lines of Bos taurus. The QTL regions for the growth traits identified using a haplotype association analysis, which included the gene-specific SNP markers for both genes in this study, were in agreement with previous studies. The gene-specific SNP marker association analysis indicated that the SNP in myf5 had a significant additive effect on PWADG in the M1 line of Beefbooster Inc. (P < 0.10), and a significant additive effect (P < 0.05) and a significant dominance effect (P < 0.10) on ADGF in the M3 line of Beefbooster Inc. When the data from the two commercial lines were pooled, the SNP in myf5 showed a significant association with PWADG (P < 0.10) and with ADGF (P < 0.05). The association between the SNP and BWT, however, did not reach a significance level in the M1 line, the M3 line, or across the lines. For igf1, no significant association between the SNP and the growth traits was detected in either the M1 line or the M3 line, whereas there was only a significant dominance effect (P < 0.10) on BWT detected for the SNP in igfl when the data from the two commercial lines were pooled. These results suggest that myf5 is a strong candidate gene that influences PWADG and ADGF in beef cattle. The SNP of igf1 may not be a causative or close to the causative mutation that affects the three growth traits in the populations of beef cattle examined in this study. Other SNP of igf1 and myf5 or other genes in their respective chromosomal regions, however, should also be studied.

Induction of GADD gene expression by phenethylisothiocyanate in human colon adenocarcinoma cells

Phenethylisothiocyanate (PEITC), a potential cancer chemopreventive agent, induces colon cancer cell death, but the mechanism is not entirely clear. Therefore, the aim of this study was to further clarify the molecular effects of PEITC in causing death of human colon adenocarcinoma cells. When incubated with PEITC, HCT-116 colonocytes showed morphological features characteristic of apoptosis, such as irregular cell shape, translocation of plasma membrane phosphatidylserine, and also chromatin condensation and fragmentation. These changes occurred after single-strand breaks in DNA were detected, suggesting that PEITC induced irreparable DNA damage, which in turn triggered the process of apoptosis. DNA macroarray analysis of a selected small cluster of apoptosis-related genes revealed noticeably higher expression of only GADD45, which was confirmed by gene-specific relative RT-PCR analysis. This led to investigation of other GADD gene members possibly affected by PEITC. Whereas GADD34 mRNA expression increased just slightly, there was an appreciable elevation of the mRNA for GADD153, which is recognized as a pro-apoptotic gene. The effect of PEITC on GADD153 was attenuated by either actinomycin D or N-acetylcysteine, suggesting that PEITC-induced upregulation of GADD153 mRNA expression was partly at the level of transcriptional activation involving reactive oxygen species. Additionally, PEITC-induced upregulation of GADD153 mRNA expression did not appear to require p53, based on the observation that PEITC also increased GADD153 mRNA expression in HCT-15 colonocytes, which are known to express mutant p53. These findings suggest that PEITC creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn helps trigger apoptosis.

Clinical heterogeneity and molecular findings in five Polish patients with glycerol kinase deficiency: investigation of two splice site mutations with computerized splice junction analysis and Xp21 gene-specific mRNA analysis

Five cases of glycerol kinase deficiency are presented with clinical, biochemical, and genetic results. Two had the glycerol kinase deficiency as part of an Xp21 contiguous gene deletion syndrome—complex form—and three had an isolated form of the enzyme deficiency. In these we found two splice site mutations (IVS1 + 4A > G, IVS9-1G > T) and one insertion (1393_1394insG). In patients with the complex form, a deletion of the DAX1, GK genes and the distal part of the DMD gene was found. A computerized study was performed to predict the effects of the splice site mutations. It showed that the IVS9-1G > T mutation substantially altered and removed the wild-type site and enhanced a cryptic site seven nucleotides downstream, and that the IVS1 + 4A > G diminished the strength of the wild-type donor site from strong to leaky. To verify these predictions, we developed an RT-PCR system with gene-specific primers that exclusively amplifies the Xp21 glycerol kinase gene transcript. Identification of individuals at risk is motivated by a need to avoid delay in a correct diagnosis. For reliable identification of heterozygotes for isolated glycerol kinase deficiency, knowledge of the specific mutation in the proband is required. This is easily obtained with the RT-PCR analyses developed in this study.

Tandemly duplicated Safener-induced glutathione S-transferase genes from Triticum tauschii contribute to genome- and organ-specific expression in hexaploid wheat.

Glutathione S-transferase (GST) gene expression was examined in several Triticum species, differing in genome constitution and ploidy level, to determine genome contribution to GST expression in cultivated, hexaploid bread wheat (Triticum aestivum). Two tandemly duplicated tau class GST genes (TtGSTU1 and TtGSTU2) were isolated from a single bacterial artificial chromosome clone in a library constructed from the diploid wheat and D genome progenitor to cultivated wheat, Triticum tauschii. The genes are very similar in genomic structure and their encoded proteins are 95% identical. Gene-specific reverse transcriptase-polymerase chain reaction analysis revealed differential transcript accumulation of TtGSTU1 and TtGSTU2 in roots and shoots. Expression of both genes was induced by herbicide safeners, 2,4-dichlorophenoxyacetic acid and abscisic acid, in the shoots of T. tauschii; however, expression of TtGSTU1 was always higher than TtGSTU2. In untreated seedlings, TtGSTU1 was expressed in both shoots and roots, whereas TtGSTU2 expression was only detected in roots. RNA gel-blot analysis of ditelosomic, aneuploid lines that are deficient for 6AS, 6BS, or 6DS chromosome arms of cultivated, hexaploid bread wheat showed differential genome contribution to safener-induced GST expression in shoots compared with roots. The GST genes from the D genome of hexaploid wheat contribute most to safener-induced expression in the shoots, whereas GSTs from the B and D genomes contribute to safener-induced expression in the roots.

Repair of 8-oxoG is slower in endogenous nuclear genes than in mitochondrial DNA and is without strand bias

DNA is vulnerable to the attack of certain oxygen radicals and one of the major DNA lesions formed is 7,8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic lesion that can mispair with adenine. The repair of 8-oxoG was studied by measuring the gene specific removal of 8-oxoG after treatment of Chinese hamster ovary (CHO) fibroblasts with the photosensitizer Ro19-8022. This compound introduces 8-oxoG lesions, which can then be detected with the Escherichia coli formamidopyrimidine DNA glycosylase (FPG). In this report we present gene specific repair analysis of endogenous genes situated in different important cellular regions and also the first analysis of strand specific DNA repair of 8-oxoG in an endogenous gene. We were not able to detect any preferential repair of transcribed genes compared to non-transcribed regions and we did not detect any strand-bias in the repair of the housekeeping gene, dihydrofolate reductase ( DHFR). In vivo, mitochondrial DNA is highly exposed to reactive oxygen species (ROS), and we find that the repair of 8-oxoG is more efficient in the mitochondrial DNA than in the nuclear DNA.

P-glycoprotein and sister P-glycoprotein sequences from mummichog, Fundulus heteroclitus, liver and intestine

P-glycoproteins (Pgps) responsible for efflux of xenobiotics from drug-resistant and normal cells may also contribute to xenobiotic resistance in aquatic organisms. We recently reported elevation of Pgp in liver and liver tumors of mummichog ( Fundulus heteroclitus) inhabiting a creosote-contaminated site (Cooper, P. S., Van Veld, P. A., & Vogelbein, W. K. (1999). Altered expression of the xenobiotic transporter P-glycoprotein in liver and liver tumors of mummichog ( Fundulus heteroclitus) from a creosote-contaminated environment. Biomarkers, 4, 48–58). Here we provide sequence information on specific forms of Pgp in this resistant population. Reverse transcriptase-polymerase chain reaction was used to amplify overlapping cDNA fragments corresponding to the C-terminal halves of two Pgp-related sequences from liver and intestine. From liver, we amplified 3 kb of a cDNA similar to mammalian sister Pgp (Spgp). We also amplified 2.7 kb of another cDNA from mummichog intestine and liver that was similar to the mammalian Mdr Pgp. Analysis of these sequences confirms that Spgps are well conserved proteins that are closely related to but distinct from true Pgps. Features of the single mummichog Mdr sequence suggest that the initial gene duplication event producing the mammalian mdr1 and mdr2 gene lineages occurred after the separation of teleost and tetrapod vertebrates. These cDNA fragments will provide useful probes for screening fish cDNA and genomic libraries for Pgp homologs and for gene-specific analysis of Pgp expression in fish tissues.

Human immunodeficiency virus reverse transcriptase and protease sequence database.

The HIV RT and Protease Sequence Database is an online relational database that catalogs evolutionary and drug-related human immunodeficiency virus (HIV) reverse transcriptase (RT) and protease sequence variation (http://hivdb.stanford.edu). The database contains a compilation of nearly all published HIV RT and protease sequences including International Collaboration database submissions (e.g., GenBank) and sequences published in journal articles. Sequences are linked to data about the source of the sequence sample and the antiretroviral drug treatment history of the individual from whom the isolate was obtained. The database is curated and sequences are annotated with data from >230 literature references. Users can retrieve additional data and view alignments of sequence sets meeting specific criteria (e.g., treatment history, subtype, presence of a particular mutation). A gene-specific sequence analysis program, new user-defined queries and nearly 2000 additional sequences were added in 1999.

The Trp64Arg polymorphism of the beta 3-adrenergic receptor gene is not associated with obesity or type 2 diabetes mellitus in a large population-based Caucasian cohort.

The beta3-adrenergic receptor (3-BAR) is assumed to play a role in the regulation of energy balance by increasing lipolysis and thermogenesis. A recently detected allelic polymorphism (Trp64Arg polymorphism) has been suggested to contribute to the development of obesity and non-insulin-dependent diabetes mellitus. We examined the prevalence of the two 3-BAR alleles in Germany and looked for associations between 3-BAR genotype and metabolic disorders (obesity and type 2 diabetes mellitus). From over 6450 participants in the Diabetomobile Study, a nationwide epidemiologic study on the prevalence of metabolic disorders (carried out from 1993 to 1996 in Germany), 1259 participants were randomly chosen. The 3-BAR genotype status was determined by 3-BAR gene-specific genomic PCR and consecutive restriction fragment length polymorphism analysis. The frequencies of the different genotypes in the examined cohort were as follows: Trp64/Trp64, 88.3%; Trp64/Arg64, 10.8%; and Arg64/ Arg64, 0.8%. No significant differences between the different genotypes were found when comparing age, body mass index, weight, total and high-density lipoprotein (HDL) cholesterol, fasting insulin, HbA11, and blood pressure; neither did the type 2 diabetes mellitus participants in the different genotype groups differ significantly in terms of age of diabetes onset or HbA11. This is the largest population-based study on the Trp64Arg polymorphism reported yet. The Arg64 allele of the 3-BAR gene was found commonly in Germany. In our cohort, no significant associations between the Arg64 allele and metabolic disorders (e.g. obesity, type 2 diabetes mellitus, dyslipidemia, or hypertension) were detected.

Three subsets of genes whose tissue specific expression is sex and age-dependent can be identified within the rat alpha 2u-globulin family.

The rat alpha 2u-globulins are encoded by a multigene family whose 20-25 members are subjected to multihormonal regulation that is dependent upon the sex of the animal, the developmental stage and the tissue being examined. Using RT-PCR and diagnostic restriction analysis of the products, we have examined the specificity of the expression of different members of the gene family. All family members can be classified into three subsets, depending on how the amplified cDNA responds to digestion with ApaLI, SstI and VspI. Subset A contains the restriction sites for both ApaLI and SstI but not VspI and typifies the genes expressed in the salivary glands of both mature and juvenile animals of both sexes, where it is the only subset expressed. This subset of genes also accounts for all the transcripts observed in the kidneys and mammary glands of juvenile males. Although subset A was represented in the transcript populations of all the other tissues examined, its proportion relative to the total varied greatly. The two other subsets were subset V, which contains only the restriction site for VspI, and subset N, which lacks all three restriction sites. In all the other tissues examined, two or all three of the subsets were expressed, usually in a manner that was unique to the sex and age of the tissue in question. The proportion of each of the three alpha 2u-globulin subsets in the alpha 2u-globulin gene family was determined by quantitation of the restriction products of amplified genomic DNA. Interestingly, the most prevalent subset in the genome (N) has the most limited tissue expression pattern, but is found in liver and preputial glands, the tissues expressing the most substantial quantities of alpha 2u-globulin. These results indicate the complexity of the regulation of the alpha 2u-globulins and point to the necessity for gene specific analyses if the expression of the family is to be understood in molecular terms.

Three subsets of genes whose tissue specific expression is sex and age‐dependent can be identified within the rat α2u‐globulin family

The rat α2u-globulins are encoded by a multigene family whose 20–25 members are subjected to multihormonal regulation that is dependent upon the sex of the animal, the developmental stage and the tissue being examined. Using RT-PCR and diagnostic restriction analysis of the products, we have examined the specificity of the expression of different members of the gene family. All family members can be classified into three subsets, depending on how the amplified cDNA responds to digestion with ApaLI, SstI and VspI. Subset A contains the restriction sites for both ApaLI and SstI but not VspI and typifies the genes expressed in the salivary glands of both mature and juvenile animals of both sexes, where it is the only subset expressed. This subset of genes also accounts for all the transcripts observed in the kidneys and mammary glands of juvenile males. Although subset A was represented in the transcript populations of all the other tissues examined, its proportion relative to the total varied greatly. The two other subsets were subset V, which contains only the restriction site for VspI, and subset N, which lacks all three restriction sites. In all the other tissues examined, two or all three of the subsets were expressed, usually in a manner that was unique to the sex and age of the tissue in question. The proportion of each of the three α2u-globulin subsets in the α2u-globulin gene family was determined by quantitation of the restriction products of amplified genomic DNA. Interestingly, the most prevalent subset in the genome (N) has the most limited tissue expression pattern, but is found in liver and preputial glands, the tissues expressing the most substantial quantities of α2u-globulin. These results indicate the complexity of the regulation of the α2u-globulins and point to the necessity for gene specific analyses if the expression of the family is to be understood in molecular terms. Dev. Genet. 21:234–244, 1997. © 1997 Wiley-Liss, Inc.

The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments

Here, a complete study is described of all the genes and isoenzymes for aspartate aminotransferase (AspAT) present in Arabidopsis thaliana. Four classes of cDNAs representing four distinct AspAT genes (ASP1-ASP4) have been cloned from Arabidopsis. Sequence analysis of the cDNAs suggests that the encoded proteins are targeted to different subcellular compartments. ASP1 encodes a mitochondrial form of AspAT, ASP3 encodes a chloroplastic/plastidic form of AspAT, whereas ASP2 and ASP4 each encode cytosolic forms of AspAT. Three distinct AspAT holoenzymes (AAT1-AAT3) were resolved by activity gel analysis. Organelle isolation reveals that AAT1 is mitochondrial-localized, AAT3 is plastid-localized, and AAT2 is cytosolic. Gene-specific Northern analysis reveals that each Asp mRNA accumulates differentially with respect to organ-type. However, the individual Asp mRNAs show no dramatic fluctuations in response to environmental stimuli such as light. Southern analysis reveals that four distinct nuclear genes probably represent the entire AspAT gene family in Arabidopsis. These molecular studies shed light on the subcellular synthesis of aspartate in Arabidopsis and suggest that some of the AspAT isoenzymes may play overlapping roles in plant nitrogen metabolism.

PhyB is evolutionarily conserved and constitutively expressed in rice seedling shoots

Southern blot analysis indicates that the rice genome contains single copies of genes encoding type A (phyA) and type B (phyB) phytochromes. We have isolated overlapping cDNA and genomic clones encoding the entire phyB polypeptide. This monocot sequence is more closely related to phyB from the dicot, Arabidopsis (73% amino acid sequence identity), than it is to the phyA gene in the rice genome (50% identity). These data support the proposal that phyA and phyB subfamilies diverged early in plant evolution and that subsequent divergence accompanied the evolution of monocots and dicots. Moreover, since rice and Arabidopsis phyB polypeptides are more closely related to one another (73% identity) than are monocot and dicot phyA sequences (63-65% identity), it appears that phyB has evolved more slowly than phyA. Sequence conservation between phyA and phyB is greatest in a central core region surrounding the chromophore attachment site, and least toward the amino-terminal and carboxy-terminal ends of the polypeptides, although hydropathy analysis suggests that the overall structure of the two phytochromes has been conserved. Gene-specific Northern blot analysis indicates that, whereas phyA is negatively regulated by phytochrome in rice seedling shoots in the manner typical of monocots, phyB is constitutively expressed irrespective of light treatment. In consequence, phyA and phyB transcripts are equally abundant in fully green tissue. Since Arabidopsis phyB mRNA levels are also unaffected by light, the present results suggest that this mode of regulation is evolutionarily conserved among phyB genes, perhaps reflecting differences in the functional roles of the different phytochrome subfamilies.

Dark-induced and organ-specific expression of two asparagine synthetase genes in Pisum sativum.

Nucleotide sequence analysis of cDNAs for asparagine synthetase (AS) of Pisum sativum has uncovered two distinct AS mRNAs (AS1 and AS2) encoding polypeptides that are highly homologous to the human AS enzyme. The amino-terminal residues of both AS1 and AS2 polypeptides are identical to the glutamine-binding domain of the human AS enzyme, indicating that the full-length AS1 and AS2 cDNAs encode glutamine-dependent AS enzymes. Analysis of nuclear DNA shows that AS1 and AS2 are each encoded by single genes in P.sativum. Gene-specific Northern blot analysis reveals that dark treatment induces high-level accumulation of AS1 mRNA in leaves, while light treatment represses this effect as much as 30-fold. Moreover, the dark-induced accumulation of AS1 mRNA was shown to be a phytochrome-mediated response. Both AS1 and AS2 mRNAs also accumulate to high levels in cotyledons of germinating seedlings and in nitrogen-fixing root nodules. These patterns of AS gene expression correlate well with the physiological role of asparagine as a nitrogen transport amino acid during plant development.

Developmentally Regulated Expression of the Gene Family for Cytosolic Glutamine Synthetase in Pisum sativum

In Pisum sativum, two classes of genes encode distinct isoforms of cytosolic glutamine synthetase (GS). The first class comprises two nearly identical or "twin" GS genes (GS341 and GS132), while the second comprises a single GS gene (GS299) distinct in both coding and noncoding regions from the "twin" GS genes. Gene-specific analyses were used to monitor the individual contribution of each gene for cytosolic GS during root nodule development and in cotyledons during germination, two contexts where large amounts of ammonia must be assimilated by GS for nitrogen transport. mRNAs corresponding to all three genes for cytosolic GS were shown to accumulate coordinately during a time course of nodule development. All the GS mRNAs also accumulate to wild-type levels in mutant nodules formed by a nifD(-) strain of Rhizobium leguminosarum indicating that induced GS expression in pea root nodules does not depend on the production of ammonia. Distinct patterns of expression for the two classes of GS genes were observed in certain mutant root nodules and most dramatically in cotyledons of germinating seedlings. The different patterns of expression between the two classes of genes for cytosolic GS suggests that their distinct gene products may serve nonoverlapping functions during pea development.