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PubMed2001

Mapping and characterization of the mouse and human SS18 genes, two human SS18-like genes and a mouse Ss18 pseudogene.

We have previously isolated and characterized a mouse cDNA orthologous to the human synovial sarcoma associated SS18 (formerly named SSXT and SYT) cDNA. Here, we report the characterization of the genomic structure of the mouse Ss18 gene. Through in silico methods with sequence information contained in the public databases, we did the same for the human SS18 gene and two human SS18 homologous genes, SS18L1 and SS18L2. In addition, we identified a mouse Ss18 processed pseudogene and mapped it to chromosome 1, band A2-3. The mouse Ss18 gene, which is subject to extensive alternative splicing, is made up of 11 exons, spread out over approximately 45 kb of genomic sequence. The human SS18 gene is also composed of 11 exons with similar intron-exon boundaries, spreading out over about 70 kb of genomic sequence. One alternatively spliced exon, which is not included in the published SS18 cDNA, corresponds to a stretch of sequence which we previously identified in the mouse Ss18 cDNA. The human SS18L1 gene, which is also made up of 11 exons with similar intron-exon boundaries, was mapped to chromosome 20 band q13.3. The smaller SS18L2 gene, which is composed of three exons with similar boundaries as the first three exons of the other three genes, was mapped to chromosome 3 band p21. Through sequence and mutation analyses this gene could be excluded as a candidate gene for 3p21-associated renal cell cancer. In addition, we created a detailed BAC map around the human SS18 gene, placing it unequivocally between the CA-repeat marker AFMc014wf9 and the dihydrofolate reductase pseudogene DHFRP1. The next gene in this map, located distal to SS18, was found to be the TBP associated factor TAFII-105 (TAF2C2). Further analogies between the mouse Ss18 gene, the human SS18 gene and its two homologous genes were found in the putative promoter fragments. All four promoters resemble the promoters of housekeeping genes in that they are TATA-less and embedded in canonical CpG islands, thus explaining the high and widespread expression of the SS18 genes.

Alternative Splicing↗
PubMed2005

(TG/CA)n repeats in human gene families: abundance and selective patterns of distribution according to function and gene length.

BACKGROUND: Creation of human gene families was facilitated significantly by gene duplication and diversification. The (TG/CA)n repeats exhibit length variability, display genome-wide distribution, and are abundant in the human genome. Accumulation of evidences for their multiple functional roles including regulation of transcription and stimulation of recombination and splicing elect them as functional elements. Here, we report analysis of the distribution of (TG/CA)n repeats in human gene families. RESULTS: The 1,317 human gene families were classified into six functional classes. Distribution of (TG/CA)n repeats were analyzed both from a global perspective and from a stratified perspective based on their biological properties. The number of genes with repeats decreased with increasing repeat length and several genes (53%) had repeats of multiple types in various combinations. Repeats were positively associated with the class of Signaling and communication whereas, they were negatively associated with the classes of Immune and related functions and of Information. The proportion of genes with (TG/CA)n repeats in each class was proportional to the corresponding average gene length. The repeat distribution pattern in large gene families generally mirrored the global distribution pattern but differed particularly for Collagen gene family, which was rich in repeats. The position and flanking sequences of the repeats of Collagen genes showed high conservation in the Chimpanzee genome. However the majority of these repeats displayed length polymorphism. CONCLUSION: Positive association of repeats with genes of Signaling and communication points to their role in modulation of transcription. Negative association of repeats in genes of Information relates to the smaller gene length, higher expression and fundamental role in cellular physiology. In genes of Immune and related functions negative association of repeats perhaps relates to the smaller gene length and the directional nature of the recombinogenic processes to generate immune diversity. Thus, multiple factors including gene length, function and directionality of recombinogenic processes steered the observed distribution of (TG/CA)n repeats. Furthermore, the distribution of repeat patterns is consistent with the current model that long repeats tend to contract more than expand whereas, the reverse dynamics operates in short repeats.

Animals↗
PubMed1993

Glycophorin B and glycophorin E genes arose from the glycophorin A ancestral gene via two duplications during primate evolution.

Human glycophorin A, B, and E genes are homologous from the 5'-flanking region to 1 kilobase downstream from the exon encoding the transmembrane region. Analysis of human Alu sequences at the transition site from the homologous to nonhomologous region suggested that the GPA gene most closely resembles the ancestral gene, whereas GPB and GPE genes arose by homologous recombination within the Alu repetitive sequence, and acquired 3' sequences from an unrelated gene (Kudo, S., and Fukuda, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4619-4623; Kudo, S., and Fukuda, M. (1990) J. Biol. Chem. 265, 1102-1110). To understand glycophorin gene evolution in primate phylogeny, transmembrane and Alu regions of several primate genomes were amplified by the polymerase chain reaction and their sequences were analyzed. These studies revealed that the GPA gene was present in all primates studied, and the GPB gene was present in pygmy chimpanzee, chimpanzee, and gorilla, but absent from orangutan and gibbon. GPE gene was present in all species with a GPB gene, but was detected in only 7 out of 16 gorillas. The 24-base pair insertion sequence found in the transmembrane exon of the human GPE gene was shown to be derived from the ancestral GPB gene and was inserted into the ancestral GPE gene prior to gorilla divergence. The recombination site in the GPA gene was confirmed to be within an Alu repetitive sequence. We conclude that GPB and GPE genes arose from an ancestral GPA gene via two gene duplications occurring during primate evolution, prior to gorilla divergence.

Amino Acid Sequence↗
PubMed1996

Amplification of a DEAD box gene (DDX1) with the MYCN gene in neuroblastomas as a result of cosegregation of sequences flanking the MYCN locus.

A DEAD box gene (DDX1) characterized by a motif with a putative RNA helicase was found at elevated levels, with multiple copies, in a neuroblastoma and in some retinoblastoma cell lines in which the MYCN gene was amplified. The present study was aimed at determining whether amplification of the DDX1 gene is critical for human neuroblastomas exhibiting MYCN gene amplification. Extended DNA panels of tumors and cell lines revealed amplification of the DDX1 gene in approximately half of the specimens exhibiting MYCN gene amplification, which is in good agreement with a finding reported recently. Because its profile was similar to that of the cDNA marker G21 and another flanking DNA marker, clone 8, both of which localize outside the core of the amplicon of the MYCN gene, we noted that we could localize the DDX1 gene in relation to the MYCN gene. Utilizing pulsed-field gel electrophoresis according to a method based on the combinatorial alignment of multiple single digests and a 5.5-megabase map surrounding the MYCN locus, we mapped the DDX1 gene within a 100 kb region about 400 kb upstream from the MYCN gene, where G21 is localized. Further hybridization experiments with both genes, complete sequencing of G21, and its comparison with that of the DDX1 gene eventually confirmed that the DDX1 gene is identical to G21. G21 is a cDNA clone isolated by differential screening of a library from a neuroblastoma cell line, IMR-32, but its function has not yet been identified. Coamplification of the DDX1 gene with the MYCN gene is a consequence of the segregation of continuous DNA stretches spanning both loci during the amplification process.

Amino Acid Sequence↗
PubMed1997

The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation.

We examined the expression of Zic1, Zic2, and Zic3 genes in the mouse embryo by means of in situ hybridization. Zic genes were found as a group of genes coding for zinc finger proteins that are expressed in a restricted manner in the adult mouse cerebellum. We showed that the genes are the vertebrate homologues of Drosophila odd-paired, which may play an essential role in parasegmental subdivision and in visceral mesoderm development. The expression of the three Zic genes was first detected at gastrulation in a spatially restricted manner. At neurulation, the expression became restricted to the dorsal neural ectoderm and dorsal paraxial mesoderm. During organogenesis, the three genes were expressed in specific regions of several developing organs, including dorsal areas of the brain, spinal cord, paraxial mesenchyme, and epidermis, the marginal zone of the neural retina and distal regions of the developing limb. For all stages, significant differences in the spatial expression of Zic1, Zic2, and Zic3 were observed. Furthermore, the expression of Zic genes in Pax3, Wnt-1, and Wnt-3a mutant embryos suggested that Zic genes are not primarily regulated by the three genes which were expressed in dorsal areas similar to Zic genes. However, in open brain, a mutant with severe neural tube defects, and in the Wnt-3a mutant mice, the expression of Zic genes was changed. The changed expression pattern in Wnt-3a mutant mice suggests that Zic genes in the neural tube are regulated by the factors from notochord. Our findings suggest that Zic genes are involved in many developmental processes. Furthermore, analysis of gene expression patterns in different mouse mutants indicated that Zic genes may act upstream of many known developmental regulatory genes.

Animals↗
PubMed1997

Analysis of the Rb gene and cyclin-dependent kinase 4 inhibitor genes (p16INK4 and p15INK4B) in human ovarian carcinoma cell lines.

In the present study, we analyzed human ovarian carcinoma cell lines for abnormalities in the tumor suppressor gene Rb (retinoblastoma) and in cyclin-dependent kinase 4 (CDK4) inhibitor genes (p16INK4 and p15INK4B) using molecular biology techniques. For the Rb gene, in all six cell lines (PA-1, Caov-3 and -4, OVCAR-3, SK-OV-3, and Kuramochi), Rb gene abnormality was not detected using Southern blotting. In the Caov-3 cell line transcripts were not detectable by either Northern blot or polymerase chain reaction. Sequence analysis of the entire coding region of the Rb gene revealed point mutations (AAC to GAC) resulting in codon 123 (Asn to Asp) changes in the Caov-4 cell line. In the PA-1 cell line both wild-type Rb and mutant-type Rb (codon 798: CGG to TGG) were expressed, and in the OVCAR-3 cell line both wild-type Rb and mutant-type Rb (codon 704: ATG to GTG) were expressed. In four of six human ovarian carcinoma cell lines Rb gene abnormality was detected. For the p16INK4 and p15INK4B genes, only the SK-OV-3 cell line had abnormalities. There was a gene rearrangement or minor deletion of the p16INK4 gene in the SK-OV-3 cell line, while the p15INK4B gene was deleted in this cell line. In the SK-OV-3 cell line no mRNAs of p16INK4 and p15INK4B were expressed. At the point of Rb gene inactivation, we can explain five cell lines of six: four cell lines had abnormalities in the Rb gene itself, which is another mechanism by which the Rb gene is inactivated, while one cell line (SK-OV-3) had abnormalities in CDK4 inhibitor genes, another of the inactivation mechanisms of the Rb gene. These data suggest that abnormalities of Rb and CDK4 inhibitor genes (p16INK4, p15INK4B) may be involved in human ovarian carcinogenesis.

Carcinoma↗
PubMed1996

Multiple gene copies for bombyxin, an insulin-related peptide of the silkmoth Bombyx mori: structural signs for gene rearrangement and duplication responsible for generation of multiple molecular forms of bombyxin.

Thirty-eight genes that encode bombyxin, the insulin-related brain secretory peptide of the silkmoth Bombyx mori, have been cloned and characterized. These genes have been classified into four families, A, B, C and D, according to their sequence similarity. All the bombyxin genes lack introns. Five of them have structural features of pseudogenes. The 38 genes cluster in the three DNA segments of Bombyx in unique distribution patterns. Their arrangement has been classified into three categories: gene pairs, gene triplets and single genes. In the pairs, two bombyxin genes belonging to families B and A (B/A) or to families B and C (B/C) are apposed with opposite transcriptional orientation. All triplets are arranged in the order of the family-B, family-C and family-A genes, and the transcriptional directions of the family-C and family-A genes are opposite to the direction of the family-B gene. The bombyxin gene triplets may have been generated by an unequal crossing-over between two gene pairs, B/A and B/C. Crossing-over may have occurred in the bombyxin family-B genes to increase their structural diversity. Duplications may have served to multiply the bombyxin gene triplets. These genomic rearrangements are thought to have led to the generation of multiple bombyxin gene copies and their diversity in structure and genomic organization.

Amino Acid Sequence↗
PubMed2006

Gene expression and gene therapy imaging.

The fast growing field of molecular imaging has achieved major advances in imaging gene expression, an important element of gene therapy. Gene expression imaging is based on specific probes or contrast agents that allow either direct or indirect spatio-temporal evaluation of gene expression. Direct evaluation is possible with, for example, contrast agents that bind directly to a specific target (e.g., receptor). Indirect evaluation may be achieved by using specific substrate probes for a target enzyme. The use of marker genes, also called reporter genes, is an essential element of MI approaches for gene expression in gene therapy. The marker gene may not have a therapeutic role itself, but by coupling the marker gene to a therapeutic gene, expression of the marker gene reports on the expression of the therapeutic gene. Nuclear medicine and optical approaches are highly sensitive (detection of probes in the picomolar range), whereas MRI and ultrasound imaging are less sensitive and require amplification techniques and/or accumulation of contrast agents in enlarged contrast particles. Recently developed MI techniques are particularly relevant for gene therapy. Amongst these are the possibility to track gene therapy vectors such as stem cells, and the techniques that allow spatiotemporal control of gene expression by non-invasive heating (with MRI guided focused ultrasound) and the use of temperature sensitive promoters.

Animals↗
PubMed2006

Single-copy genes define a conserved order between rice and wheat for understanding differences caused by duplication, deletion, and transposition of genes.

The high-quality rice genome sequence is serving as a reference for comparative genome analysis in crop plants, especially cereals. However, early comparisons with bread wheat showed complex patterns of conserved synteny (gene content) and colinearity (gene order). Here, we show the presence of ancient duplicated segments in the progenitor of wheat, which were first identified in the rice genome. We also show that single-copy (SC) rice genes, those representing unique matches with wheat expressed sequence tag (EST) unigene contigs in the whole rice genome, show more than twice the proportion of genes mapping to syntenic wheat chromosome as compared to the multicopy (MC) or duplicated rice genes. While 58.7% of the 1,244 mapped SC rice genes were located in single syntenic wheat chromosome groups, the remaining 41.3% were distributed randomly to the other six non-syntenic wheat groups. This could only be explained by a background dispersal of genes in the genome through transposition or other unknown mechanism. The breakdown of rice-wheat synteny due to such transpositions was much greater near the wheat centromeres. Furthermore, the SC rice genes revealed a conserved primordial gene order that gives clues to the origin of rice and wheat chromosomes from a common ancestor through polyploidy, aneuploidy, centromeric fusions, and translocations. Apart from the bin-mapped wheat EST contigs, we also compared 56,298 predicted rice genes with 39,813 wheat EST contigs assembled from 409,765 EST sequences and identified 7,241 SC rice gene homologs of wheat. Based on the conserved colinearity of 1,063 mapped SC rice genes across the bins of individual wheat chromosomes, we predicted the wheat bin location of 6,178 unmapped SC rice gene homologs and validated the location of 213 of these in the telomeric bins of 21 wheat chromosomes with 35.4% initial success. This opens up the possibility of directed mapping of a large number of conserved SC rice gene homologs in wheat. Overall, only 46.4% of these SC genes code for proteins with known functional domains; the remaining 53.6% have unknown function, and hence, represent an important, but yet, under explored category of genes.

Gene Deletion↗
PubMed1982

Structure of human immunoglobulin gamma genes: implications for evolution of a gene family.

We have cloned five human immunoglobulin gamma genes from a fetal liver gene library. Four of them encode the known human immunoglobulin gamma chains gamma 1, gamma 2, gamma 3 and gamma 4. A fifth gamma gene seems to be a pseudogene. Nucleotide sequence determination demonstrates that the gamma 3 gene contains four separate hinge exons. Comparison of these hinge exons with those of the other gamma genes indicates that the first hinge exon is homologous to that of the pseudogene, and that the other three hinge exons are homologous to that of the gamma 1 gene, suggesting that the gamma 3 gene ancestor is a hybrid gene created by unequal crossing-over between the ancestral gamma 1 and psi gamma genes. Amplification of the gamma 1-type hinge exon probably followed to complete the gamma 3 gene. This hypothesis inevitably postulates the gene order 5'-gamma 1-gamma 3-psi gamma-3'. Cloning of overlapping chromosomal segments demonstrates that the gamma 2 gene is located 19 kb 5' to the gamma 4 gene. These analyses indicate that the human gamma-gene family has evolved by several types of DNA rearrangemet, including duplication of a complete gene; duplication of a hinge exon; and reassortment of exons by unequal cross-over between two adjacent genes.

Animals↗
PubMed1998

The ParaHox gene cluster is an evolutionary sister of the Hox gene cluster.

Genes of the Hox cluster are restricted to the animal kingdom and play a central role in axial patterning in divergent animal phyla. Despite its evolutionary and developmental significance, the origin of the Hox gene cluster is obscure. The consensus is that a primordial Hox cluster arose by tandem gene duplication close to animal origins. Several homeobox genes with high sequence identity to Hox genes are found outside the Hox cluster and are known as 'dispersed' Hox-like genes; these genes may have been transposed away from an expanding cluster. Here we show that three of these dispersed homeobox genes form a novel gene cluster in the cephalochordate amphioxus. We argue that this 'ParaHox' gene cluster is an ancient paralogue (evolutionary sister) of the Hox gene cluster; the two gene clusters arose by duplication of a ProtoHox gene cluster. Furthermore, we show that amphioxus ParaHox genes have co-linear developmental expression patterns in anterior, middle and posterior tissues. We propose that the origin of distinct Hox and ParaHox genes by gene-cluster duplication facilitated an increase in body complexity during the Cambrian explosion.

Amino Acid Sequence↗
PubMed1999

Attenuated expression of the serum responsive T1 gene in ras transformed fibroblasts due to the inhibition of c-fos gene activity.

The T1 gene encodes a protein, which shares homology with the IL-1 receptors. In fibroblasts, T1 is induced by growth factors and in response to the onset of oncogene expression. The c-fos gene is transiently activated in these situations and was shown to be the major mediator of T1 gene induction. In contrast, the sustained expression of a ras oncogene in NIH3T3 cells resulted in the downregulation of basal T1 gene activity and the attenuation of T1 gene induction in response to mitogenic signals. Likewise, the immediate early genes encoding c-Fos, FosB, and Fra-2 are repressed in these cells. T1 gene repression could be overcome by the forced expression of c-fos in ras transformed fibroblasts. Thus, the lack of c-fos gene expression is the likely cause for ras mediated T1 gene repression. Fra-1, in contrast to the other three members of the Fos family, is permanently synthesized in high amounts in ras transformed NIH3T3 fibroblasts. We show that AP-1, which is abundant in these cells throughout the whole cell cycle, consists predominantly of Fra-1/c-Jun and Fra1/JunD heterodimers. We provide evidence that Fra1/c-Jun heterodimers are responsible for the repression of c-fos gene induction following serum stimulation. The introduction of a dominant negative version of c-Jun into ras transformed fibroblasts was able to rescue c-fos gene induction in response to serum stimulation, further demonstrating that AP-1 is indeed involved in c-fos gene repression. We conclude that oncogenic ras mediates the activation of the fra-1 gene which results in elevated AP-1 activity throughout the cell cycle. Fra-1 containing AP-1 complexes repress the c-fos and possibly other immediate early genes thereby preventing the induction of certain delayed early genes such as the T1 gene in response to mitogenic stimulation.

3T3 Cells↗
PubMed2004

Gene expression analysis of the pro-oestrous-stage rat uterus reveals neuroligin 2 as a novel steroid-regulated gene.

In the present study, differential gene expression in the uteri of ovariectomised (OVX) and pro-oestrous rats (OVX v. pro-oestrus pair) was investigated using cDNA expression array analysis. Differential uterine gene expression in OVX rats and progesterone (P(4))-injected OVX rats (OVX v. OVX + P(4) pair) was also examined. The uterine gene expression profiles of these two sets of animals were also compared for the effects of P(4) treatment. RNA samples were extracted from uterine tissues and reverse transcribed in the presence of [alpha(32)P]-dATP. Membrane sets of rat arrays were hybridised with cDNA probe sets. Northern blot analysis was used to validate the relative gene expression patterns obtained from the cDNA array. Of the 1176 cDNAs examined, 23 genes showed significant (>two-fold) changes in expression in the OVX v. pro-oestrus pair. Twenty of these genes were upregulated during pro-oestrus compared with their expression in the OVX rat uterus. In the OVX v. OVX + P(4) pair, 22 genes showed significant (>two-fold) changes in gene expression. Twenty of these genes were upregulated in the OVX + P(4) animals. The genes for nuclear factor I-XI, afadin, neuroligin 2, semaphorin Z, calpain 4, cyclase-associated protein homologue, thymosin beta-4X and p8 were significantly upregulated in the uteri of the pro-oestrus and OVX + P(4) rats of both experimental pairs compared with the OVX rat uteri. These genes appear to be under the control of P(4). One of the most interesting findings of the present study is the unexpected and marked expression of the neuroligin 2 gene in the rat uterus. This gene is expressed at high levels in the central nervous system and acts as a nerve cell adhesion factor. According to Northern blot analysis, neuroligin 2 gene expression was higher during the pro-oestrus and metoestrus stages than during the oestrus and dioestrus stages of the oestrous cycle. In addition, neuroligin 2 mRNA levels were increased by both 17beta-oestradiol (E(2)) and P(4), although P(4) administration upregulated gene expression to a greater extent than injection of E(2). These results indicate that neuroligin 2 gene expression in the rat uterus is under the control of both E(2) and P(4), which are secreted periodically during the oestrous cycle.

Animals↗
PubMed2001

Dual-tagging gene trap of novel genes in Drosophila melanogaster.

A gene-trap system is established for Drosophila. Unlike the conventional enhancer-trap system, the gene-trap system allows the recovery only of fly lines whose genes are inactivated by a P-element insertion, i.e., mutants. In the gene-trap system, the reporter gene expression reflects precisely the spatial and temporal expression pattern of the trapped gene. Flies in which gene trap occurred are identified by a two-step screening process using two independent markers, mini-w and Gal4, each indicating the integration of the vector downstream of the promoter of a gene (dual tagging). mini-w has its own promoter but lacks a polyadenylation signal. Therefore, mini-w mRNA is transcribed from its own promoter regardless of the vector integration site in the genome. However, the eyes of flies are not orange or red unless the vector is incorporated into a gene enabling mini-w to be spliced to a downstream exon of the host gene and polyadenylated at the 3' end. The promoter-less Gal4 reporter is expressed as a fusion mRNA only when it is integrated downstream of the promoter of a host gene. The exons of trapped genes can be readily cloned by vectorette RT-PCR, followed by RACE and PCR using cDNA libraries. Thus, the dual-tagging gene-trap system provides a means for (i) efficient mutagenesis, (ii) unequivocal identification of genes responsible for mutant phenotypes, (iii) precise detection of expression patterns of trapped genes, and (iv) rapid cloning of trapped genes.

Animals↗
PubMed2006

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Genome-wide search of gene conversions in duplicated genes of mouse and rat.

Gene conversion is considered to play important roles in the formation of genomic makeup such as homogenization of multigene families and diversification of alleles. We devised two statistical tests on quartets for detecting gene conversion events. Each "quartet" consists of two pairs of orthologous sequences supposed to have been generated by a duplication event and a subsequent speciation of two closely related species. As example data, EnsEMBL mouse and rat cDNA sequences were used to obtain a genome-wide picture of gene conversion events. We extensively sampled 2,641 quartets that appear to have resulted from duplications after the divergence of primates and rodents and before mouse-rat speciation. Combination of our new tests with Sawyer's and Takahata's tests enhanced the detection sensitivity while keeping false positives as few as possible. About 18% (488 quartets) were shown to be highly positive for gene conversion using this combined test. Out of them, 340 (13% of the total) showed signs of gene conversion in mouse sequence pairs. Those gene conversion-positive gene pairs are mostly linked in the same chromosomes, with the proportion of positive pairs in the linked and unlinked categories being 15% and 1%, respectively. Statistical analyses showed that (1) the susceptibility to gene conversion correlates negatively with the physical distance, especially the frequency of 29% was observed for gene pairs whose distances are smaller than 55 kb; (2) the occurrence of gene conversions does not depend on the transcriptional direction; (3) small gene families consisting of between three and six contiguous genes are highly prone to gene conversion; and (4) frequency of gene conversions greatly varies depending on functional categories, and cadherins favor gene conversion, while vomeronasal receptors type 1 and immunoglobulin V-type proteins disfavor it. These findings will be useful to deepen the understanding of the roles of gene conversion.

Alleles↗
PubMed1999

Glyceraldehyde-3-phosphate dehydrogenase gene diversity in eubacteria and eukaryotes: evidence for intra- and inter-kingdom gene transfer.

Cyanobacteria contain up to three highly divergent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes: gap1, gap2, and gap3. Genes gap1 and gap2 are closely related at the sequence level to the nuclear genes encoding cytosolic and chloroplast GAPDH of higher plants and have recently been shown to play distinct key roles in catabolic and anabolic carbon flow, respectively, of the unicellular cyanobacterium Synechocystis sp. PCC6803. In the present study, sequences of 10 GAPDH genes distributed across the cyanobacteria Prochloron didemni, Gloeobacter violaceus PCC7421, and Synechococcus PCC7942 and the alpha-proteobacterium Paracoccus denitrificans and the beta-proteobacterium Ralstonia solanacearum were determined. Prochloron didemni possesses homologs to the gap2 and gap3 genes from Anabaena, Gloeobacter harbors gap1 and gap2 homologs, and Synechococcus possesses gap1, gap2, and gap3. Paracoccus harbors two highly divergent gap genes that are related to gap3, and Ralstonia possesses a homolog of the gap1 gene. Phylogenetic analyses of these sequences in the context of other eubacterial and eukaryotic GAPDH genes reveal that divergence across eubacterial gap1, and gap2, and gap3 genes is greater than that between eubacterial gap1 and eukaroytic glycolytic GapC or between eubacterial gap2 and eukaryotic Calvin cycle GapAB. These data strongly support previous analyses which suggested that eukaryotes acquired their nuclear genes for GapC and GapAB via endosymbiotic gene transfer from the antecedents of mitochondria and chloroplasts, and extend the known range of sequence diversity of the antecedent eubacterial genes. Analyses of available GAPDH sequences from other eubacterial sources indicate that the glycosomal gap gene from trypanosomes (cytosolic in Euglena) and the gap gene from the spirochete Treponema pallidum are each other's closest relatives. This specific relationship can therefore not reflect organismal evolution but must be the result of an interkingdom gene transfer, the direction of which cannot be determined with certainty at present. Contrary to this, the origin of the cytosolic Gap gene from trypanosomes can now be clearly defined as gamma-proteobacterial, since the newly established Ralstonia sequence (beta-proteobacteria) branches basally to the gamma-proteobacterial/trypanosomal assemblage.

Amino Acid Sequence↗
PubMedDate not supplied

Azorhizobium caulinodans nitrogen fixation (nif/fix) gene regulation: mutagenesis of the nifA -24/-12 promoter element, characterization of a ntrA(rpoN) gene, and derivation of a model.

Using site-directed mutagenesis, mutations were introduced in the -24/-12 promoter element of the Azorhizobium caulinodans nifA gene, and chimeric nifA-lacZ reporter gene fusions were constructed. Single base-pair mutations in the conserved -25 or -13 G residues were found to reduce or abolish nifA promoter activity, respectively, demonstrating that the -24/-12 promoter element is important for nifA gene expression and suggesting the involvement of a sigma 54 (NtrA/RpoN)-type transcription factor in nifA gene regulation. A 2-bp mutation at positions -25 and -16 was found to create a relatively nitrogen-control-independent, highly expressed nifA promoter. Using a heterologous ntrA(rpoN) gene probe, an A. caulinodans ntrA(rpoN)-like gene was cloned and the DNA sequence of this gene and flanking regions was determined. The presence of three open reading frames (ORF1-3) was demonstrated. ORF2 was found to contain regions sharing a high degree of homology with all characterized bacterial ntrA(rpoN) genes. ORF1 was found to share homology with ORFs found upstream of other bacterial ntrA(rpoN) genes, which have been postulated to encode members of a superfamily of ATP-binding proteins. Transposon Tn5 insertion mutations were introduced into the cloned ntrA(rpoN) gene, and chromosomal ntrA(rpoN)::Tn5 A. caulinodans mutants were created. The resulting mutants were found to be unable to fix nitrogen in the free-living state (Nif- in culture) or in stem or root nodules induced on Sesbania rostrata (Fix- in planta), and to be unable to grow aerobically in the presence of nitrate as sole nitrogen source (Ntr-). A nifH-lacZ gene fusion was found to be silent in ntrA(rpoN)::Tn5 mutant strains, but a nifA-lacZ gene fusion was found to be expressed at a wild-type level, suggesting that the ntrA(rpoN) gene identified here controls the expression of some of the A. caulinodans nif genes, but not the central nif regulatory gene nifA. Based on these results, a new model for the regulation of nif/fix gene expression in A. caulinodans is proposed.

Amino Acid Sequence↗
PubMed1994

Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae.

We have developed a large-scale screen to identify genes expressed at different times during the life cycle of Saccharomyces cerevisiae and to determine the subcellular locations of many of the encoded gene products. Diploid yeast strains containing random lacZ insertions throughout the genome have been constructed by transformation with a mutagenized genomic library. Twenty-eight hundred transformants containing fusion genes expressed during vegetative growth and 55 transformants containing meiotically induced fusion genes have been identified. Based on the frequency of transformed strains producing beta-galactosidase, we estimate that 80-86% of the yeast genome (excluding the rDNA) contains open reading frames expressed in vegetative cells and that there are 93-135 meiotically induced genes. Indirect immunofluorescence analysis of 2373 strains carrying fusion genes expressed in vegetative cells has identified 245 fusion proteins that localize to discrete locations in the cell, including the nucleus, mitochondria, endoplasmic reticulum, cytoplasmic dots, spindle pole body, and microtubules. The DNA sequence adjacent to the lacZ gene has been determined for 91 vegetative fusion genes whose products have been localized and for 43 meiotically induced fusions. Although most fusions represent genes unidentified previously, many correspond to known genes, including some whose expression has not been studied previously and whose products have not been localized. For example, Sec21-beta-gal fusion proteins yield a Golgi-like staining pattern, Ty1-beta-gal fusion proteins localize to cytoplasmic dots, and the meiosis-specific Mek1/Mre4-beta-gal and Spo11-beta-gal fusion proteins reside in the nucleus. The phenotypes in haploid cells have been analyzed for 59 strains containing chromosomal fusion genes expressed during vegetative growth; 9 strains fail to form colonies indicating that the disrupted genes are essential. Fifteen additional strains display slow growth or are impaired for growth on specific media or in the presence of inhibitors. Of 39 meiotically induced fusion genes examined, 14 disruptions confer defects in spore formation or spore viability in homozygous diploids. Our results will allow researchers who identify a yeast gene to determine immediately whether that gene is expressed at a specific time during the life cycle and whether its gene product localizes to a specific subcellular location.

Base Sequence↗