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PubMed1988

Evolutionary dynamics of tryptophan tRNAs in Mycoplasma capricolum.

Mycoplasma capricolum uses two tryptophan codons, the "universal" nonsense codon UGA and the universal codon UGG. The bacterium contains two tryptophan tRNAs, one with anticodon UCA, (U: 2'-O-methyl U derivative), and the other with CCA (5'-C: partially 2'-O-methylated). tRNAUCA would translate codons UGA and probably UGG by wobbling. tRNACCA is much less charged by tryptophan in the cells than tRNAUCA, and the intracellular amount of tRNACCA is 5-10 times lower than that of tRNAUCA. The genes for these two tRNAs are separated by a terminator-like structure in a single operon. In vitro transcription experiments suggest that the predominance of tRNAUCA over tRNACCA results from the attenuation of transcription by this terminator-like structure.

Base Sequence↗
PubMed1995

Double-stranded RNA in rice: a novel RNA replicon in plants.

The entire sequence of 13952 nucleotides of a plasmid-like, double-stranded RNA (dsRNA) from rice was assembled from more than 50 independent cDNA clones. The 5' non-coding region of the coding (sense) strand spans over 166 nucleotides, followed by one long open reading frame (ORF) of 13716 nucleotides that encodes a large putative polyprotein of 4572 amino acid residues, and by a 70-nucleotide 3' non-coding region. This ORF is apparently the longest reported to date in the plant kingdom. Amino acid sequence comparisons revealed that the large putative polyprotein includes an RNA helicase-like domain and an RNA-dependent RNA polymerase (replicase)-like domain. Comparisons of the amino acid sequences of these two domains and of the entire genetic organization of the rice dsRNA with those found in potyviruses and the CHV1-713 dsRNA of chestnut blight fungus suggest that the rice dsRNA is located evolutionarily between potyviruses and the CHV1-713 dsRNA. This plasmid-like dsRNA in rice seems to constitute a novel RNA replicon in plants.

Amino Acid Sequence↗
PubMed1989

The progenitor of ATP synthases was closely related to the current vacuolar H+-ATPase.

The gene encoding the proteolipid of the vacuolar H+-ATPase of yeast was cloned and sequenced. The deduced amino acid sequence of the yeast protein is highly homologous to that of the proteolipid from bovine chromaffin granules. In contrast to other membrane proteins the transmembrane segments of the bovine and yeast proteolipids were much more conserved than the hydrophilic parts. The fourth transmembrane segment, which contains the DCCD-binding site, was conserved 100%. Comparison of vacuolar and eubacterial proteolipids revealed a homology which pointed to a common ancestral gene that underwent gene duplication to form the vacuolar proteolipids. Additional support for this notion came from the amino acid sequences of subunits involved in the catalytic sectors of archaebacterial ATP synthase and plant and yeast vacuolar H+-ATPases, which reveal extensive sequence homology. Slight, but significant, homology between the archaebacterial and eubacterial ATP synthases was observed. These observations might suggest that the progenitor of ATP synthases was closely related to the present vacuolar H+-ATPases.

Amino Acid Sequence↗
PubMed1989

Cloning and analysis of the 5' portion of the human type-III procollagen gene (COL3A1).

We have isolated overlapping clones containing the 5'-terminal portion of the human pro-alpha 1(III) collagen gene (COL3A1). This has enabled us to extend our previous studies and thus generate a restriction map of nearly 64 kb of DNA encompassing all of COL3A1 and more than 20 kb of flanking sequences. Aside from the complete nucleotide and amino acid sequences of type-III N-pre-propeptide, this study has established the number of the corresponding exons, whose relative organization deviates from the pattern observed in the analogous regions of type-I procollagen genes, COL1A1 and COL1A2. Moreover, we have sequenced 1628 bp of the 5'-flanking region of COL3A1, from the transcription start point (tsp) to an AluI repetitive element. Pairwise comparison with the analogous segment of the mouse gene has showed 78% sequence similarity in nearly 270 bp immediately preceding the tsp and including the TATA element and a presumptive NF-1 binding site. Relatively close to the tsp, but upstream from the region of homology with the murine gene, a potential AP-1 binding site has also been identified.

Amino Acid Sequence↗
PubMed2004

Genome data mining of lactic acid bacteria: the impact of bioinformatics.

Lactic acid bacteria (LAB) have been widely used in food fermentations and, more recently, as probiotics in health-promoting food products. Genome sequencing and functional genomics studies of a variety of LAB are now rapidly providing insights into their diversity and evolution and revealing the molecular basis for important traits such as flavor formation, sugar metabolism, stress response, adaptation and interactions. Bioinformatics plays a key role in handling, integrating and analyzing the flood of 'omics' data being generated. Reconstruction of metabolic potential using bioinformatics tools and databases, followed by targeted experimental verification and exploration of the metabolic and regulatory network properties, are the present challenges that should lead to improved exploitation of these versatile food bacteria.

Adaptation, Biological↗
PubMed2004

Evolution of bacterial diversity and the origins of modularity.

A characteristic feature of all organisms is modular organisation: the tendency for groups of genes to interact in such a way as to limit the extent of pleiotropic effects among characters belonging to different functional complexes. While the implications of modularity for the evolution of variability have been much discussed the evolutionary origins remain obscure. Here we develop a model, with special reference to signal transduction cascades of bacteria, which predicts that in the face of ecological opportunity and lateral gene transfer, selection will favour modular genome architectures because such architectures minimise the pleiotropic effects associated with accommodation of potentially beneficial foreign DNA.

Base Composition↗
PubMed1991

Deduced amino acid sequence of Escherichia coli adenosine deaminase reveals evolutionarily conserved amino acid residues: implications for catalytic function.

The goal of the research reported here is to identify evolutionarily conserved amino acid residues associated with enzymatic deamination of adenosine. To do this, we isolated molecular clones of the Escherichia coli adenosine deaminase gene by functional complementation of adenosine deaminase deficient bacteria and deduced the amino acid sequence of the enzyme from the nucleotide sequence of the gene. Nucleotide sequence analysis revealed the presence of a 996-nucleotide open reading frame encoding a protein of 332 amino acids having a molecular weight of 36,345. The deduced amino acid sequence of the E. coli enzyme has approximately 33% identity with those of the mammalian adenosine deaminases. With conservative amino acid substitutions the overall sequence homology approaches 50%, suggesting that the structures and functions of the mammalian and bacterial enzymes are similar. Additional amino acid sequence analysis revealed specific residues that are conserved among all three adenosine deaminases and four AMP deaminases for which sequence information is currently available. In view of previously published enzymological data and the conserved amino acid residues identified in this study, we propose a model to account for the enzyme-catalyzed hydrolytic deamination of adenosine. Potential catalytic roles are assigned to the conserved His 214, Cys 262, Asp 295, and Asp 296 residues of mammalian adenosine deaminases and the corresponding conserved amino acid residues in bacterial adenosine deaminase and the eukaryotic AMP deaminases.

Adenosine Deaminase↗
PubMed1992

Molecular characterization of cDNA encoding for adenylate kinase of rice (Oryza sativa L.).

Two types of genes (Adk-a, and Adk-b) encoding for adenylate kinase (AK, EC 2.7.4.3.) were isolated from the cDNA library constructed from poly(A)+ RNA of rice (Oryza sativa L.). Two cDNAs were heterogeneous at 5' and 3' ends of non-coding sequences and had possible polyadenylation signals. One of the genes, Adk-a, had 1154 bp sequences encoding 241 amino acid residues, while the other type, Adk-b, contained 1085 bp sequences encoding for 243 amino acid residues. Homology between Adk-a and Adk-b was 73.7% in nucleotide sequences, and 90.8% in amino acid level. Two genes showed about 53% homology to bovine mitochondrial adenylate kinase (AK2) at nucleotide and amino acid levels. Concerning the codon usage of rice AK genes, T was abundant at the third position of a codon in the reading frames. In order to examine the enzyme activity of the protein encoded by the rice cDNA, Adk-a was cloned into an expression vector, pUC119, which was introduced into Escherichia coli strain CV2, a temperature-sensitive mutant of adenylate kinase. We found that the transformant carrying the rice Adk-a gene in the sense orientation recovered cell growth at non-permissive high temperature (42 degrees C) and expressed enzyme activities higher than the untransformed CV2 and the transformant possessing Adk-a cDNA in the antisense orientation. These observations suggest that rice Adk-a codes a biologically active enzyme. Furthermore, sucrose was found to regulate the transcription of AK genes in rice cell cultures. Organ related accumulation of mRNA in whole plants was also found.

Adenylate Kinase↗
PubMed2004

Splicing of U12-type introns deposits an exon junction complex competent to induce nonsense-mediated mRNA decay.

Metazoan cells have two pathways for intron removal involving the U2- and U12-type spliceosomes, which contain mostly nonoverlapping sets of small nuclear ribonucleoproteins. We show that in vitro splicing of a U12-type intron assembles an exon junction complex (EJC) that is comparably positioned and contains many of the same components as that deposited by the U2-type spliceosome. The presence of a U12-type intron downstream of a premature termination codon within an open reading frame (ORF) induces nonsense-mediated decay of the mRNA in vivo. These findings suggest a common pathway for EJC assembly by the two spliceosomes and highlight the evolutionary age of the EJC and its downstream functions in gene expression.

Cell Line↗
PubMed2003

The integrity of a cholesterol-binding pocket in Niemann-Pick C2 protein is necessary to control lysosome cholesterol levels.

The neurodegenerative disease Niemann-Pick Type C2 (NPC2) results from mutations in the NPC2 (HE1) gene that cause abnormally high cholesterol accumulation in cells. We find that purified NPC2, a secreted soluble protein, binds cholesterol specifically with a much higher affinity (K(d) = 30-50 nM) than previously reported. Genetic and biochemical studies identified single amino acid changes that prevent both cholesterol binding and the restoration of normal cholesterol levels in mutant cells. The amino acids that affect cholesterol binding surround a hydrophobic pocket in the NPC2 protein structure, identifying a candidate sterol-binding location. On the basis of evolutionary analysis and mutagenesis, three other regions of the NPC2 protein emerged as important, including one required for efficient secretion.

Amino Acid Sequence↗
PubMed1990

Chimeric phage-bacterial enzymes: a clue to the modular evolution of genes.

Pneumococcal peptidoglycan amidase (N-acetylmuramoyl-L-alanine amidase, EC 3.5.1.28) and phage CPL1 lysozyme degrade a common substrate (choline-containing pneumococcal cell walls); the former hydrolyzes the bond between muramic acid and alanine, whereas the latter breaks down the linkage between muramic acid and glucosamine. The amino acid sequences of their C-terminal domains are homologous. Chimeric genes were constructed by site-directed mutagenesis: a unique SnaBI restriction site in the cpl1 gene, coding for the phage lysozyme, was introduced at a location equivalent to the SnaBI site present in the lytA gene, which codes for the pneumococcal amidase. The resulting genes expressed lytic activities at levels similar to those of the parental genes. The gene products, which have been purified to electrophoretical homogeneity, exhibited unusual combined biochemical properties--e.g., by exchange of protein domains, we have switched the regulatory properties of these enzymes without altering their catalytic activities. Chimeric gene construction in Streptococcus pneumoniae and its bacteriophages is an excellent model to study the modular organization of genes and proteins and to help to establish evolutionary relationships between phage and bacteria. These constructions provide an experimental approach to the molecular processes involved in cassette recruitment during evolution and contribute support to the concept of bacteria as adaptable chimeras.

Amino Acid Sequence↗
PubMed1984

Composite human VK genes and a model of their evolution.

A phage library and two cosmid libraries were screened for human VK genes. Two recombinant phage and four cosmid clones were analysed in detail by restriction mapping and sequencing. Each one contained a single VKI sequence. Two of these six sequences are potentially functional VK genes and four are pseudogenes. Two pseudogenes derived from different genomic DNAs are highly homologous and are therefore either allelic variants or the products of a recent duplication event. Comparisons of our sequences with all fully determined human VKI amino acid and DNA sequences reveal identical segments which at first sight appear like minigenes. But these segments do not coincide with the subregions and some of the segments include both, framework and complementarity determining regions (FR, CDR, ref. 2). The findings may be explained by an evolutionary model generating composite genes by gene conversion and selection.

Alleles↗
PubMed1995

Novel protein families in archaean genomes.

In a quest for novel functions in archaea, all archaean hypothetical open reading frames (ORFs), as annotated in the Swiss-Prot protein sequence database, were used to search the latest databases for the identification of characterized homologues. Of the 95 hypothetical archaean ORFs, 25 were found to be homologous to another hypothetical archaean ORF, while 36 were homologous to non-archaean proteins, of which as many as 30 were homologous to a characterized protein family. Thus the level of sequence similarity in this set reaches 64%, while the level of function assignment is only 32%. Of the ORFs with predicted functions, 12 homologies are reported here for the first time and represent nine new functions and one gene duplication at an acetyl-coA synthetase locus. The novel functions include components of the transcriptional and translational apparatus, such as ribosomal proteins, modification enzymes and a translation initiation factor. In addition, new enzymes are identified in archaea, such as cobyric acid synthase, dCTP deaminase and the first archaean homologues of a new subclass of ATP binding proteins found in fungi. Finally, it is shown that the putative laminin receptor family of eukaryotes and an archaean homologue belong to the previously characterized ribosomal protein family S2 from eubacteria. From the present and previous work, the major implication is that archaea seem to have a mode of expression of genetic information rather similar to eukaryotes, while eubacteria may have proceeded into unique ways of transcription and translation. In addition, with the detection of proteins in various metabolic and genetic processes in archaea, we can further predict the presence of additional proteins involved in these processes.

Animal Population Groups↗
PubMed1990

Functional and evolutionary relatedness of genes for exopolysaccharide synthesis in Rhizobium meliloti and Rhizobium sp. strain NGR234.

Rhizobium meliloti SU47 and Rhizobium sp. strain NGR234 produce distinct exopolysaccharides that have some similarities in structure. R. meliloti has a narrow host range, whereas Rhizobium strain NGR234 has a very broad host range. In cross-species complementation and hybridization experiments, we found that several of the genes required for the production of the two polysaccharides were functionally interchangeable and similar in evolutionary origin. NGR234 exoC and exoY corresponded to R. meliloti exoB and exoF, respectively. NGR234 exoD was found to be an operon that included genes equivalent to exoM, exoA, and exoL in R. meliloti. Complementation of R. meliloti exoP, -N, and -G by NGR234 R'3222 indicated that additional equivalent genes remain to be found on the R-prime. We were not able to complement NGR234 exoB with R. meliloti DNA. In addition to functional and evolutionary equivalence of individual genes, the general organization of the exo regions was similar between the two species. It is likely that the same ancestral genes were used in the evolution of both exopolysaccharide biosynthetic pathways and probably of pathways in other species as well.

Biological Evolution↗
PubMed1991

Sequence analysis of the clpG gene, which codes for surface antigen CS31A subunit: evidence of an evolutionary relationship between CS31A, K88, and F41 subunit genes.

The clpG gene coding for the CS31A subunit was localized on a 0.9-kb SphI fragment from the recombinant plasmid pAG315. This was established by testing the ability of subclones to hybridize with a 17-meric oligonucleotide probe obtained from N-terminal analysis of the CS31A subunit. The nucleotide sequence of the region coding for CS31A was determined. From primer extension analysis, two initiation translation start sites were detected. Two possible promoterlike sequences were identified; the ribosome binding site and the translation terminator are proposed. Inverted repeat sequences leading to the formation of possible hairpin structures of the transcripts were found on the 5' untranslated region of clpG. The deduced amino acid composition was in close agreement with the chemical amino acid composition and sequence match with the first 25 N-terminal amino acids from the published N-terminal sequence of the purified CS31A subunit. The clpG gene codes for a mature protein of 257 amino acids with a molecular size of 26,777 Da. An obvious homology was observed when the amino acid sequence of CS31A was compared with those of K88 and F41. This homology includes five different conserved sequences of up to 19 identical amino acids, which is associated with conserved proline. An extensive change in the CS31A region homologous to that identified to contain the K88 receptor binding site might be responsible for the functional divergence between CS31A and K88.

Amino Acid Sequence↗
PubMed2001

The TFIID components human TAF(II)140 and Drosophila BIP2 (TAF(II)155) are novel metazoan homologues of yeast TAF(II)47 containing a histone fold and a PHD finger.

The RNA polymerase II transcription factor TFIID comprises the TATA binding protein (TBP) and a set of TBP-associated factors (TAF(II)s). TFIID has been extensively characterized for yeast, Drosophila, and humans, demonstrating a high degree of conservation of both the amino acid sequences of the constituent TAF(II)s and overall molecular organization. In recent years, it has been assumed that all the metazoan TAF(II)s have been identified, yet no metazoan homologues of yeast TAF(II)47 (yTAF(II)47) and yTAF(II)65 are known. Both of these yTAF(II)s contain a histone fold domain (HFD) which selectively heterodimerizes with that of yTAF(II)25. We have cloned a novel mouse protein, TAF(II)140, containing an HFD and a plant homeodomain (PHD) finger, which we demonstrated by immunoprecipitation to be a mammalian TFIID component. TAF(II)140 shows extensive sequence similarity to Drosophila BIP2 (dBIP2) (dTAF(II)155), which we also show to be a component of Drosophila TFIID. These proteins are metazoan homologues of yTAF(II)47 as their HFDs selectively heterodimerize with dTAF(II)24 and human TAF(II)30, metazoan homologues of yTAF(II)25. We further show that yTAF(II)65 shares two domains with the Drosophila Prodos protein, a recently described potential dTAF(II). These conserved domains are critical for yTAF(II)65 function in vivo. Our results therefore identify metazoan homologues of yTAF(II)47 and yTAF(II)65.

Amino Acid Motifs↗
PubMed2001

Interpretation of X chromosome dose at Sex-lethal requires non-E-box sites for the basic helix-loop-helix proteins SISB and daughterless.

For Drosophila melanogaster flies, sexual fate is determined by the X chromosome number. The basic helix-loop-helix protein product of the X-linked sisterlessB (sisB or scute) gene is a key indicator of the X dose and functions to activate the switch gene Sex-lethal (Sxl) in female (XX), but not in male (XY), embryos. Zygotically expressed sisB and maternal daughterless (da) proteins are known to form heterodimers that bind E-box sites and activate transcription. We examined SISB-Da binding at Sxl by using footprinting and gel mobility shift assays and found that SISB-Da binds numerous clustered sites in the establishment promoter Sxl(Pe). Surprisingly, most SISB-Da sites at Sxl(Pe) differ from the canonical CANNTG E-box motif. These noncanonical sites have 6-bp CA(G/C)CCG and 7-bp CA(G/C)CTTG cores and exhibit a range of binding affinities. We show that the noncanonical sites can mediate SISB-Da-activated transcription in cell culture. P-element transformation experiments show that these noncanonical sites are essential for Sxl(Pe) activity in embryos. Together with previous deletion analysis, the data suggest that the number, affinity, and position of SISB-Da sites may all be important for the operation of the Sxl(Pe) switch. Comparisons with other dose-sensitive promoters suggest that threshold responses to diverse biological signals have common molecular mechanisms, with important variations tailored to suit particular functional requirements.

Amino Acid Motifs↗