The complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli K-12.

In this report we present the complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli. This operon contains five genes encoding four of the five enzymes required for the biosynthesis of isoleucine and valine. We identify and describe the coding regions for these five structural genes and the structural and functional features of the flanking and internal regulatory regions of this operon. This new information contributes to a more complete understanding of the overall control of the biosynthesis of isoleucine and valine.     

On the tetraploid origin of the maize genome

Data from cytological and genetic mapping studies suggest that maize arose as a tetraploid. Two previous studies investigating the most likely mode of maize origin arrived at different conclusions. Gaut and Doebley [7] proposed a segmental allotetraploid origin of the maize genome and estimated that the two maize progenitors diverged at 20.5 million years ago (mya). In a similar study, using larger data set, Brendel and colleagues (quoted in [8]) suggested a single genome duplication at 16 mya. One of the key components of such analyses is to examine sequence divergence among strictly orthologous genes. In order to identify such genes, Lai and colleagues [10] sequenced five duplicated chromosomal regions from the maize genome and the orthologous counterparts from the sorghum genome. They also identified the orthologous regions in rice. Using positional information of genetic components, they identified 11 orthologous genes across the two duplicated regions of maize, and the sorghum and rice regions. Swigonova et al. [12] analyzed the 11 orthologues, and showed that all five maize chromosomal regions duplicated at the same time, supporting a tetraploid origin of maize, and that the two maize progenitors diverged from each other at about the same time as each of them diverged from sorghum, about 11.9 mya.     

Multiple-copy cluster-type organization and evolution of genes encoding O-methyltransferases in the apple

Plant O-methyltransferases (OMTs) play important roles in secondary metabolism. Two clusters of genes coding for caffeic acid OMT (COMT) have been identified in the apple genome. Three genes from one cluster and two genes from another cluster were isolated. These five genes encoding COMT, designated Mdomt1-Mdomt5 (GenBank accession nos. DQ886018-DQ886022), were distinguished by a (CT)(n) microsatellite in the 5'-UTR and two transposon-like sequences present in the promoter region and intron 1, respectively. The transposon-like sequence in intron 1 unambiguously traced the five Mdomt genes in the apple to a common ancestor. The ancestor must have undergone an initial duplication generating two progenitors, and this was followed by further duplication of these progenitors resulting in the two clusters identified in this study. The distal regions of the transposon-like sequences in promoter regions of Mdomt genes are capable of forming palindromic hairpin-like structures. The hairpin formation is likely responsible for nucleotide sequence differences observed in the promoter regions of these genes as it plays a destabilizing role in eukaryotic chromosomes. In addition, the possible mechanism of amplification of Mdomt genes in the apple genome is also discussed.     

Gene conversions in the growth hormone gene family of primates: stronger homogenizing effects in the Hominidae lineage

In humans, the growth hormone/chorionic somatomammotropin gene family is composed of five highly similar genes. We characterized the gene conversions that occurred between the growth hormone genes of 11 primate species. We detected 48 conversions using GENECONV and others were only detected using phylogenetic analyses. Gene conversions were detected in all species analyzed, their average size (± standard deviation) is 197.8 ± 230.4 nucleotides, the size of the conversions is correlated with sequence similarity and converted regions are significantly more GC-rich than non-converted regions. Gene conversions have a stronger homogenizing effect in Hominidae genes than in other primate species. They are also less frequent in conserved gene regions and towards functionally important genes. This suggests that the high degree of sequence similarity observed between the growth hormone genes of primate species is a consequence of frequent gene conversions in gene regions which are under little selective constraints.     

Growth hormone locus expands and diverges after the separation of New and Old World Monkeys

While most mammals including the prosimians have a single copy of the growth hormone (GH) gene, anthropoids possess a cluster of GH-related genes. Throughout the evolution of the main anthropoid groups [New World Monkeys (NWM), Old World Monkeys (OWM), and apes], two features stand out of the GH loci. The first is the appearance of chorionic somatommamotropin hormone (CSH) genes within the OWM lineage and the second is the expansion of the loci intergenic regions in the OWM and apes. In relation with this loci expansion, the NWM possess intergenic regions of homogeneous lengths (3.5 kb). In contrast, heterogeneous lengths (6 and 13 kb) have been reported for species of the OWM. At the present, none of the OWM genomic GH loci organizations have been described. Here, we report the genomic organization of the GH locus in the rhesus monkey, this locus has six GH-related genes separated by five intergenic regions. The 5' end gene (GH-1) encodes for the pituitary GH and is followed by CSH-1, GH-2, CSH-2, CSH-3 and CSH-4 genes. The five intergenic regions have heterogeneous lengths and also present more or less the same Alu distribution as the human GH locus. To analyze the events that contributed to the extension of the intergenic regions of the GH locus and the emergence of the regulatory elements, the five GH locus intergenic regions of the spider monkey (NWM) were sequenced. The results of comparing the loci from both species suggest that the long intergenic regions (13 kb) of the rhesus GH locus share a common ancestor with the 3.5 kb intergenic regions of the spider monkey. However, the observed increased length of the former is due to an insertion (approximately 8.7 kb) at their 3' end. Interestingly in this insert, we discovered a DNA element resembling the enhancer of the CSH genes of the human GH locus. On the other hand, we observed that the short intergenic regions (6 kb) increased by a different recombination event.     

Characterisation of the subtelomeric regions of Giardia lamblia genome isolate WBC6

Giardia trophozoites are polyploid and have five chromosomes. The chromosome homologues demonstrate considerable size heterogeneity due to variation in the subtelomeric regions. We used clones from the genome project with telomeric sequence at one end to identify six subtelomeric regions in addition to previously identified subtelomeric regions, to study the telomeric arrangement of the chromosomes. The subtelomeric regions included two retroposons, one retroposon pseudogene, and two vsp genes, in addition to the previously identified subtelomeric regions that include ribosomal DNA repeats. The presence of vsp genes in a subtelomeric region suggests that telomeric rearrangements may contribute to the generation of vsp diversity. These studies of the subtelomeric regions of Giardia may contribute to our understanding of the factors that maintain stability, while allowing diversity in chromosome structure.     

Sequence homologies in the protamine gene family of rainbow trout

We have sequenced five different rainbow trout protamine genes plus their flanking regions. The genes are not clustered and do not contain intervening sequences. There is an extremely high degree of sequence conservation in the coding and 3' untranslated regions of the gene. Downstream sequences exhibit little homology though conserved regions are found 250 base pairs 3' to the gene. There are four regions upstream of the gene that are highly conserved in the six clones, including the canonical Goldberg - Hogness box which is 45 base pairs 5' to the coding region. A second homologous region is found 90 bases upstream. Although in the same approximate location as the CAAT box found upstream of other genes, it does not contain the canonical CAAT sequence. Further upstream of the protamine genes at -115 there is an A-T rich sequence while a 25 base pair conserved sequence is located 150 bases upstream. In addition we report the presence of a potential Z-DNA region of predominantly A-C repeats approximately one kilobase downstream of one of the genes.     

Cloning and sequence comparison of the mouse, human, and chicken engrailed genes reveal potential functional domains and regulatory regions.

We have isolated and characterized genomic DNA clones for the human and chicken homologues of the mouse En-1 and En-2 genes and determined the genomic structure and predicted protein sequences of both En genes in all three species. Comparison of these vertebrate En sequences with the Xenopus En-2 [Hemmati-Brivanlou et al., 1991) and invertebrate engrailed-like genes showed that the two previously identified highly conserved regions within the En protein ]reviewed in Joyner and Hanks, 1991] can be divided into five distinct subregions, designated EH1 to EH5. Sequences 5' and 3' to the predicted coding regions of the vertebrate En genes were also analyzed in an attempt to identify cis-acting DNA sequences important for the regulation of En gene expression. Considerable sequence similarity was found between the mouse and human homologues both within the putative 5' and 3' untranslated as well as 5' flanking regions. Between the mouse and Xenopus En-2 genes, shorter stretches of sequence similarity were found within the 3' untranslated region. The 5' untranslated regions of the mouse, chicken and Xenopus En-2 genes, however, showed no similarly conserved stretches. In a preliminary analysis of the expression pattern of the human En genes, En-2 protein and RNA were detected in the embryonic and adult cerebellum respectively and not in other tissues tested. These patterns are analogous to those seen in other vertebrates. Taken together these results further strengthen the suggestion that En gene function and regulation has been conserved throughout vertebrate evolution and, along with the five highly conserved regions within the En protein, raise an interesting question about the presence of conserved genetic pathways.     

Comparative genome analysis of the yellow fever mosquito Aedes aegypti with Drosophila melanogaster and the malaria vector mosquito Anopheles gambiae

An in silico comparative genomics approach was used to identify putative orthologs to genetically mapped genes from the mosquito, Aedes aegypti, in the Drosophila melanogaster and Anopheles gambiae genome databases. Comparative chromosome positions of 73 D. melanogaster orthologs indicated significant deviations from a random distribution across each of the five A. aegypti chromosomal regions, suggesting that some ancestral chromosome elements have been conserved. However, the two genomes also reflect extensive reshuffling within and between chromosomal regions. Comparative chromosome positions of A. gambiae orthologs indicate unequivocally that A. aegypti chromosome regions share extensive homology to the five A. gambiae chromosome arms. Whole-arm or near-whole-arm homology was contradicted with only two genes among the 75 A. aegypti genes for which orthologs to A. gambiae were identified. The two genomes contain large conserved chromosome segments that generally correspond to break/fusion events and a reciprocal translocation with extensive paracentric inversions evident within. Only very tightly linked genes are likely to retain conserved linear orders within chromosome segments. The D. melanogaster and A. gambiae genome databases therefore offer limited potential for comparative positional gene determinations among even closely related dipterans, indicating the necessity for additional genome sequencing projects with other dipteran species.     

Retroviral and pseudogene insertion sites reveal the lineage of human salivary and pancreatic amylase genes from a single gene during primate evolution.

We have analyzed the junction regions of inserted elements within the human amylase gene complex. This complex contains five genes which are expressed at high levels either in the pancreas or in the parotid gland. The proximal 5'-flanking regions of these genes contain two inserted elements. A gamma-actin pseudogene is located at a position 200 base pairs upstream of the first coding exon. All of the amylase genes contain this insert. The subsequent insertion of an endogenous retrovirus interrupted the gamma-actin pseudogene within its 3'-untranslated region. Nucleotide sequence analysis of the inserted elements associated with each of the five human amylase genes has revealed a series of molecular events during the recent history of this gene family. The data indicate that the entire gene family was generated during primate evolution from one ancestral gene copy and that the retroviral insertion activated a cryptic promoter.     

Genetic characterization of the dibenzofuran-degrading Actinobacteria carrying the dbfA1A2 gene homologues isolated from activated sludge

Thirteen dibenzofuran (DF)-utilizing bacteria carrying the DF terminal dioxygenase genes homologous to those of Terrabacter sp. strain DBF63 (dbfA1A2) were newly isolated from activated sludge samples. The amplified ribosomal DNA restriction analysis and the hybridization analyses showed that these strains were grouped into five genetically different types of bacteria. The sequence analyses of the 16S rRNA genes and the dbfA1A2 homologues from these five selected isolates revealed that the isolates belonged to the genus Rhodococcus, Terrabacter or Janibacter and that they shared 99-100% conserved dbfA1A2 homologues. We investigated the genetic organizations flanking the dbfA1A2 homologues and showed that the minimal conserved DNA region present in all five selected isolates consisted of an approximately 9.0-kb region and that their outer regions became abruptly non-homologous. Among them, Rhodococcus sp. strain DFA3 possessed not only the 9.0-kb region but also the 6.2-kb region containing dbfA1A2 homologues. Sequencing of their border regions suggested that some genetic rearrangement might have occurred with insertion sequence-like elements. Also, within their conserved regions, some insertions or deletions were observed.     

Detection of nucleolar organizer and mitochondrial DNA insertion regions based on the isochore map of Arabidopsis thaliana

Eukaryotic genomes are composed of isochores, i.e. long sequences relatively homogeneous in GC content. In this paper, the isochore structure of Arabidopsis thaliana genome has been studied using a windowless technique based on the Z curve method and intuitive curves are drawn for all the five chromosomes. Using these curves, we can calculate the GC content at any resolution, even at the base level. It is observed that all the five chromosomes are composed of several GC-rich and AT-rich regions alternatively. Usually, these regions, named 'isochore-like regions', have large fluctuations in the GC content. Five isochores with little fluctuations are also observed. Detailed analyses have been performed for these isochores. A GC-rich 'isochore-like region' and a GC-isochore in chromosome II and IV, respectively, are the nucleolar organizer regions (NORs), and genes located in the two regions prefer to use GC-ending codons. Another GC-isochore located in chromosome II is a mitochondrial DNA insertion region, the position and size of this region is precisely predicted by the current method. The amino acid usage and codon preference of genes in this organellar-to-nuclear transfer region show significant difference from other regions. Moreover, the centromeres are located in GC-rich 'isochore-like regions' in all the five chromosomes. The current method can provide a useful tool for analyzing whole genomic sequences of eukaryotes.     

Structural features of transposed human VK genes and implications for the mechanism of their transpositions.

The genes encoding the variable, joining and constant regions of human immunoglobulin light chains have been localized to the short arm of chromosome 2. However, several VK genes lie outside of the locus: a single copy cluster of five VK genes is located on chromosome 22; an isolated but amplified VkI gene is found on chromosome 1; and several isolated VkI genes are on as-yet-unidentified chromosomes other than chromosome 2. Vk genes not contained within the kappa locus are termed orphons. We have attempted to gain insight into the mechanism of transposition of both the chromosome 22 cluster and the several amplified VkI genes by searching in the kappa locus for a parent copy of the former, and by analyzing the junctions between transposed VKI-containing segments and adjacent non-amplified regions. The chromosome 22 orphon cluster must have been non-duplicatively transposed. Sequence features at the junctions of this and other orphon regions are direct and inverted repeats, and, in one case, an Alu repeat. These unusual features may have predisposed the orphon regions to transposition by serving as target sites for enzymes involved in recombination.     

DNase I-defined chromatin configuration of the human CD3 gene cluster.

The three CD3 genes on human chromosome 11q23 encode proteins (gamma, delta and epsilon) which form part of the antigen receptor on T lymphocytes. All three genes are clustered within 50 kb and are activated approximately contemporaneously during the early stages of T cell ontogeny. In order to pinpoint potential regulatory sequences important for locus activation and tissue-specific gene expression, the chromatin structure of almost 90 kb of this region has been probed in five cell lines using the endonuclease pancreatic DNase I. A set of DNase I hypersensitive (HS) sites has been defined in T cell chromatin, five of which were strong and not found in non-T cells, with the exception of the erythroleukaemia cell line K562, in which three sites were weakly expressed, correlating with a low level of delta mRNA. The subset of five HS sites map close to the CD3 genes and lie in regions which contain elements of defined function: the gamma promoter; the delta promoter and its 3' enhancer; and the epsilon promoter and its 3' enhancer. Since no further major T cell-restricted HS sites lie within the 90kb of the CD3 locus analysed, these five regions may contain all the sequences important for CD3 gene expression.     

Conserved signals in the 5' flanking region of eukaryotic nuclear tRNA genes.

The statistical analysis of 5' flanking regions of eukaryotic tRNA genes was done. The analysis of nucleotides in the sequence of fungi and invertebrates showed a high content of A and T in the flanking regions versus coding regions where G and C dominate. In contrast to these results in vertebrates sequences the preferences of any nucleotide in flanking regions was not observed. The analysis of tetrads showed five conserved signals: TTGT, (T/A)(T/A)ATA, A(C/T)(C/A)A in the tRNA genes of fungi, (A/T)TGA of invertebrates and (A/T)GAG of vertebrates. The analysis of 3' flanking regions did not show any conserved signals except well known poly-T tracks.     

Syntenic arrangements of the surface polysaccharide biosynthesis genes in Rhizobium leguminosarum

We applied a genomic approach in the identification of genes required for the biosynthesis of different polysaccharides in Rhizobium leguminosarum bv. trifolii TA1 (RtTA1). Pulsed-field gel electrophoresis analyses of undigested genomic DNA revealed that the RtTA1 genome is partitioned into a chromosome and four large plasmids. The combination of sequencing of RtTA1 library BAC clones and PCR amplification of polysaccharide genes from the RtTA1 genome led to the identification of five large regions and clusters, as well as many separate potential polysaccharide biosynthesis genes dispersed in the genome. We observed an apparent abundance of genes possibly linked to lipopolysaccharide biosynthesis. All RtTA1 polysaccharide biosynthesis regions showed a high degree of conserved synteny between R. leguminosarum bv. viciae and/or Rhizobium etli. A majority of the genes displaying a conserved order also showed high sequence identity levels.     

Molecular basis for modulated regulation of gene expression in the arginine regulon of Escherichia coli K-12.

We compare the nucleotide sequences of the regulatory regions of five genes or groups of genes of the arginine regulon of Escherichia coli K-12: argF, argI, argR, the bipolar argECBH operon and the carAB operon. All these regions harbour one or two copies of a conserved 18 bp sequence which appears to constitute the basic arginine operator sequence (ARG box). We discuss the influence of ARG box copy number, degree of dyad symmetry, base composition, and position relative to the cognate promoter site on the derepression-repression ratios of the genes of the regulon. A novel hypothesis, based on structural considerations, is also put forward to account for the absence ot attenuation control.