A homolog of interleukin-10 is encoded by the poxvirus orf virus.

A gene encoding a polypeptide with homology to interleukin-10 (IL-10) has been discovered in the genome of orf virus (OV) strain NZ2, a parapoxvirus that infects sheep, goats, and humans. The predicted polypeptide sequence shows high levels of amino acid identity to IL-10 of sheep (80%), cattle (75%), humans (67%), and mice (64%), as well as IL-10-like proteins of Epstein-Barr virus (63%) and equine herpesvirus (67%). The C-terminal region, comprising two-thirds of the OV protein, is identical to ovine IL-10, which suggests that this gene has been captured from its host sheep during the evolution of OV. The IL-10-like gene is transcribed early. Conditioned medium from COS cells transfected with a eukaryotic expression vector containing the OV IL-10-like gene showed the same biological activity as ovine IL-10 in a murine thymocyte proliferation assay. OV IL-10 is likely to be important in immune evasion by OV, since IL-10 is a multifunctional cytokine that has inhibitory effects on nonspecific immunity and Th1 effector function.     

Drosophila Melanogaster Mitochondrial DNA: Gene Organization and Evolutionary Considerations

The sequence of a 8351-nucleotide mitochondrial DNA (mtDNA) fragment has been obtained extending the knowledge of the Drosophila melanogaster mitochondrial genome to 90% of its coding region. The sequence encodes seven polypeptides, 12 tRNAs and the 3' end of the 16S rRNA and CO III genes. The gene organization is strictly conserved with respect to the Drosophila yakuba mitochondrial genome, and different from that found in mammals and Xenopus. The high A + T content of D. melanogaster mitochondrial DNA is reflected in a reiterative codon usage, with more than 90% of the codons ending in T or A, G + C rich codons being practically absent. The average level of homology between the D. melanogaster and D. yakuba sequences is very high (roughly 94%), although insertion and deletions have been detected in protein, tRNA and large ribosomal genes. The analysis of nucleotide changes reveals a similar frequency for transitions and transversions, and reflects a strong bias against G + C on both strands. The predominant type of transition is strand specific.     

Generation of recombinant parapoxviruses: non-essential genes suitable for insertion and expression of foreign genes

Orf virus (OV) is an epitheliotropic poxvirus and belongs to the genus Parapoxvirus (PPV). PPV, especially OV, is regarded as a promising candidate for an expression vector. Among available live vaccines only strain D1701 represents a highly attenuated OV strain with clearly reduced pathogenicity. Therefore, we started to identify potentially non-essential genes or regions of D1701, which might be suitable for insertion and expression of foreign genes. The present contribution reviews some of the progress using the vegf-e (homologue of the mammalian vascular endothelial growth factor) gene locus for the generation of recombinant D1701. The vegf-e gene of D1701 is dispensable for virus growth in vitro and in vivo, and represents a major virulence determinant of OV. It is shown that foreign genes can be inserted and functionally expressed in the vegf-e locus, also leading to the induction of a specific immune response in the non-permissive host. Furthermore, it is reported that adaptation to VERO cells led to the deletion of three further regions of the OV D1701 genome, which seems to be combined with additional virus attenuation in sheep. Molecular analysis of this OV D1701 variant allows the identification of new, potentially non-essential sites in the viral genome.     

A cDNA clone encoding the ADP/ATP translocase of Drosophila melanogaster shows a high degree of similarity with the mammalian ADP/ATP translocases

Summary A complementary DNA clone encoding the ADP/ATP translocase in Drosophila melanogaster has been identified. It has been shown by sequence analysis to contain a single open reading frame that encodes a polypeptide 297 amino acids long. This polypeptide shows extensive similarities to the known eukaryotic translocase polypeptides, the similarity being greatest (up to 80% identity) to the mammalian ADP/ATP translocases. In situ hybridization to polytene chromosomes of D. melanogaster with the sequence characterized in this study showed localization at a single site on the X chromosome at 9E. DNA transfer hybridization experiments suggest that more than one gene coding for the ADP/ATP translocase is present in the D. melanogaster genome.Offprint requests to: S.G. Tsitilou     

Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus.

A cDNA clone designated OV7 encodes a polypeptide that corresponds to a highly antigenic Onchocerca volvulus protein. OV7 has significant amino acid sequence homology to the cystatin superfamily of cysteine proteinase inhibitors. In this report we establish that the OV7 recombinant protein is active as a cysteine proteinase inhibitor, and we have named it onchocystatin. It contains a cystatin-like domain that inhibits the activity of cysteine proteinases at physiological concentrations. Recombinant glutathione S-transferase-OV7 (GST-OV7, 1 microM) and maltose-binding protein-OV7 (MBP-OV7, 4 microM) fusion polypeptides inhibit 50% of the enzymatic activity of the bovine cysteine proteinase cathepsin B. Neither fusion polypeptide inhibits serine or metalloproteinases activity. The Ki for GST-OV7 fusion polypeptide is 170 nM for cathepsin B and 70 pM or 25 nM for cysteine proteinases purified from a protozoan parasite Entamoeba histolytica or the free living nematode Caenorhabditis elegans, respectively. The 5' end of the OV7 clone was isolated by polymerase chain reaction and sequenced, thus extending the previous cDNA clone to 736 base pairs. This represents the complete coding sequence of the mature onchocystatin (130 amino acids). A hydrophobic leader sequence of 32 amino acids was found, indicating a possible extracellular function of the onchocerca cysteine proteinase inhibitor.     

The nucleotide sequence of an Escherichia coli operon containing genes for the tRNA(m1G)methyltransferase, the ribosomal proteins S16 and L19 and a 21-K polypeptide.

The nucleotide sequence of a 4.6-kb SalI-EcoRI DNA fragment including the trmD operon, located at min 56 on the Escherichia coli K-12 chromosome, has been determined. The trmD operon encodes four polypeptides: ribosomal protein S16 (rpsP), 21-K polypeptide (unknown function), tRNA-(m1G)methyltransferase (trmD) and ribosomal protein L19 (rplS), in that order. In addition, the 4.6-kb DNA fragment encodes a 48-K and a 16-K polypeptide of unknown functions which are not part of the trmD operon. The mol. wt. of tRNA(m1G)methyltransferase determined from the DNA sequence is 28 424. The probable locations of promoter and terminator of the trmD operon are suggested. The translational start of the trmD gene was deduced from the known NH2-terminal amino acid sequence of the purified enzyme. The intercistronic regions in the operon vary from 9 to 40 nucleotides, supporting the earlier conclusion that the four genes are co-transcribed, starting at the major promoter in front of the rpsP gene. Since it is known that ribosomal proteins are present at 8000 molecules/genome and the tRNA-(m1G)methyltransferase at only approximately 80 molecules/genome in a glucose minimal culture, some powerful regulatory device must exist in this operon to maintain this non-coordinate expression. The codon usage of the two ribosomal protein genes is similar to that of other ribosomal protein genes, i.e., high preference for the most abundant tRNA isoaccepting species. The trmD gene has a codon usage typical for a protein made in low amount in accordance with the low number of tRNA-(m1G)methyltransferase molecules found in the cell.     

Sequence divergence of an archaebacterial gene cloned from a mesophilic and a thermophilic methanogen

Summary A 1.6-kb fragment of DNA from the thermophilic, methane-producing, anaerobic archaebacteriumMethanobacterium thermoautotrophicum H has been cloned and sequenced. This DNA complements mutations in both the purE₁ and purE₂ loci ofEscherichia coli. The sequence of theM. thermoautotrophicum DNA predicts that complementation inE. coli results from the synthesis of a polypeptide with a molecular weight of 36,249. A polypeptide apparently of this molecular weight is synthesized inE. coli minicells containing recombinant plasmids that carry the cloned fragment of methanogen DNA. We have previously cloned and sequenced a purE-complementing gene from the mesophilic methanogenMethanobrevibacter smithii. The two methanogen-derived purE-complementing genes are 53% homologous and encode polypeptides that are 45% homologous in their amino acid sequences but would be 74% homologous if conservative amino acid substitutions were considered as maintaining sequence homology. The genome ofM. thermoautotrophicum has a molar G+C content of 49.7%, whereas the genome ofM. smithii is 30.6% G+C. Conservation of encoded amino acids while accommodating the very different G+C contents is accomplished by use of different codons that encode the same amino acid. The majority of base changes occur at the third codon position. The intergenic regions of the clonedM. thermoautotrophicum DNA contain sequences previously identified as ribosome binding sites and as putative methanogen promoters. Although the two purE-complementing genes are apparently derived from a common ancestor, only the gene fromM. smithii maintains a codon usage that conforms to the RNY rule.     

赤麂线粒体全基因组的序列和结构

提取赤麂细胞株总DNA,参照我们实验室已测定的同属动物小麂线粒体全基因组序列设计引物,PCR扩增、测序、拼接,获得赤麂线粒体全基因组序列并进行生物信息学分析。赤麂线粒体全基因组序列全长16354bp。定位了22个tRNA基因、2个rRNA基因、13个蛋白编码基因和1个D-loop区。赤麂与小麂及其它哺乳动物线粒体的基因组结构相同,它们的序列同源性都较高。     

Chromosomal location effects on gene sequence evolution in mammals.

BACKGROUND: Nucleotide substitution rates and G + C content vary considerably among mammalian genes. It has been proposed that the mammalian genome comprises a mosaic of regions - termed isochores - with differing G + C content. The regional variation in gene G + C content might therefore be a reflection of the isochore structure of chromosomes, but the factors influencing the variation of nucleotide substitution rate are still open to question. RESULTS: To examine whether nucleotide substitution rates and gene G + C content are influenced by the chromosomal location of genes, we compared human and murid (mouse or rat) orthologues known to belong to one of the chromosomal (autosomal) segments conserved between these species. Multiple members of gene families were excluded from the dataset. Sets of neighbouring genes were defined as those lying within 1 centiMorgan (cM) of each other on the mouse genetic map. For both synonymous substitution rates and G + C content at silent sites, neighbouring genes were found to be significantly more similar to each other than sets of genes randomly drawn from the dataset. Moreover, we demonstrated that the regional similarities in G + C content (isochores) and synonymous substitution rate were independent of each other. CONCLUSIONS: Our results provide the first substantial statistical evidence for the existence of a regional variation in the synonymous substitution rate within the mammalian genome, indicating that different chromosomal regions evolve at different rates. This regional phenomenon which shapes gene evolution could reflect the existence of 'evolutionary rate units' along the chromosome.     

Evidence that both G + C rich and G + C poor isochores are replicated early and late in the cell cycle.

Since the G + C content of a gene is correlated to that of the isochore in which it resides, and early replicating isochores are thought to be relatively G + C rich, early replicating genes should also be rich in G + C. This hypothesis is tested on a sample of 44 mammalian genes for which replication time data and sequence information are available. Early replicating genes do not appear to be more G + C rich than late replicating genes, instead there is considerable variation in the G + C content of genes replicated during both halves of S phase. These results show that both G + C rich and poor fractions of the genome are replicated early and late in the cell cycle, and suggest that isochores are not maintained by the replication of DNA sequences in compositionally biased free nucleotide pools.     

The Pseudomonas oleovorans alkBAC operon encodes two structurally related rubredoxins and an aldehyde dehydrogenase

The Pseudomonas oleovorans alkBAC operon encodes seven proteins, of which at least three are involved in alkane hydroxylase (alkBA) and alkanol dehydrogenase (alkC) activities. We have determined the nucleotide sequence of the 2.5-kilobase pair alkA region and analyzed the role of its translation products in alkane oxidation. The alkA region contains three coding sequences, encoding two related rubredoxins (alkF and alkG) of 14- and 18-kDa molecular mass and a 52-kDa aldehyde dehydrogenase (alkH). Deletion analysis indicated that neither the 14-kDa alkF gene product (rubredoxin 1) nor the amino-terminal part of the 18-kDa alkG gene product (rubredoxin 2) is required for alkane hydroxylase activity in vivo. The product of the alkH cistron restores growth of a P. oleovorans aldehyde dehydrogenase mutant on aliphatic alcohols and aldehydes. Its amino acid sequence shows considerable homology to previously characterized aldehyde dehydrogenases from mammalian and fungal origin. The nucleotide composition of the alk genes (47% G + C) differs considerably from the G + C content of the P. oleovorans genome suggesting that the alk regulon may originate from an unrelated organism.     

Complete sequence and analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae.

The complete nucleotide sequence of the plastid genome of the unicellular primitive red alga Cyanidioschyzon merolae 10D (Cyanidiophyceae) was determined. The genome is a circular DNA composed of 149,987 bp with no inverted repeats. The G + C content of this plastid genome is 37.6%. The C. merolae plastid genome contains 243 genes, which are distributed on both strands and consist of 36 RNA genes (3 rRNAs, 31 tRNAs, tmRNA, and a ribonuclease P RNA component) and 207 protein genes, including unidentified open reading frames. The striking feature of this genome is the high degree of gene compaction; it has very short intergenic distances (approximately 40% of the protein genes were overlapped) and no genes have introns. This genome encodes several genes that are rarely found in other plastid genomes. A gene encoding a subunit of sulfate transporter (cysW) is the first to be identified in a plastid genome. The cysT and cysW genes are located in the C. merolae plastid genome in series, and they probably function together with other nuclear-encoded components of the sulfate transport system. Our phylogenetic results suggest that the Cyanidiophyceae, including C. merolae, are a basal clade within the red lineage plastids.     

DNA sequence determination and functional characterization of the OCT-plasmid-encoded alkJKL genes of Pseudomonas oleovorans

The alkBFGHJKL and alkST operons encode enzymes that allow Pseudomonas putida (oleovorans) to metabolize alkanes. In this paper we report the nucleotide sequence of a 4592 bp region of the alkBFGHJKL operon encoding the AlkJ, AlkK and AlkL polypeptides. The alkJ gene encodes a protein of 59 kilodaltons. The predicted amino acid sequence shows significant homology with four flavin proteins: choline dehydrogenase, a glucose dehydrogenase and two oxidases. AlkJ is membrane-bound and converts aliphatic medium-chain-length alcohols into aldehydes. The properties of AlkJ suggest that it is linked to the electron transfer chain. AlkJ is necessary for growth on alkanes only in P. putida alcohol dehydrogenase (AlcA) mutants. AlkK is homologous to a range of proteins which act by an ATP-dependent covalent binding of AMP to their substrate. This list includes the acetate, coumarate and long-chain fatty acid CoA ligases. The alkK gene complements a fadD mutation in Escherichia coli, which shows that it indeed encodes an acyl-CoA synthetase. AlkK is a 60 kilodalton protein located in the cytoplasm. AlkL is homologous to OmpW, a Vibrio cholerae outer membrane protein of unknown function, and a hypothetical polypeptide encoded by ytt4 in E. coli. AlkL, OmpW and Ytt4 all have a signal peptide and end with a sequence characteristic of outer membrane proteins. The alkL gene product was found in the outer membrane of E. coli W3110 containing the alk-genes. The alkL gene can be deleted without a clear effect on growth rate. Its function remains unknown. The G+C content of the alkJKL genes is 45%, identical to that of the alkBFGH genes, and significantly lower than the G+C content of the OCT-plasmid and the P. putida chromosome.     

A conserved gene encoding the 57-kDa subunit of the yeast vacuolar H+-ATPase

The peripheral (catalytic) sector of vacuolar H+-ATPases contains five different polypeptides denoted as subunits A-E in order of decreasing molecular masses from 72 to 33 kDa. The gene encoding subunit B (57 kDa) of yeast vacuolar H+-ATPase was cloned on a 5-kilobase pair genomic DNA fragment and sequenced. Four open reading frames were identified in the sequenced DNA. One of them encodes a protein of 504 amino acids with a calculated Mr of 56,557. Hydropathy plot revealed no apparent transmembrane segments. Southern analysis demonstrated that a single gene encodes this polypeptide in the yeast genome. The amino acid sequence exhibits extensive identity with the homologous protein from the plant Arabidopsis (77%). This polypeptide also contains regions of homology with the alpha subunits of H+-ATPases from mitochondria, chloroplasts, and bacteria. However, less similarity was detected when it was compared with the beta subunits of those enzymes. The implication of these phenomena on the evolution of proton pumps is discussed.     

小鼠SNP与Car2、Gpi1基因多态性的关联性

目的研究单核苷酸多态性(single nucleotide polymorphism,SNP)与小鼠生化标记基因Car2、Gpi1多态性间的关联性。方法从DNA、cDNA和蛋白多肽3个方面分析研究Car2与Gpi1基因的多态性。结果 Car2基因DNA和cDNA中有3个SNP与Car2a/b多态性相关,分别为外显子2中的C(T)、G(C)和外显子7中的A(G);蛋白多肽中发现第38位Gln/His与Car2a/b多态性相关,对应于外显子2中的G(C)。Gpi1基因DNA中没有发现与Gpi1a/b多态性相关的SNP;cDNA水平有2个SNP与Gpi1a/b多态性相关,分别为外显子9中的T(C)和18中的A(G);蛋白多肽中发现第247位Phe/Leu与Gpi1a/b多态性相关,对应于外显子9中的T(C)。结论 Gln/His(38)、Phe/Leu(247)间的转换可能分别是近交系小鼠形成Car2a/b,Gpi1a/b多态性的原因。     

Organization and expression of two tandemly oriented genes encoding ribulosebisphosphate carboxylase/oxygenase activase in barley

We have isolated and structurally characterized genomic DNA and cDNA sequences encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase) activase from barley (Hordeum vulgare L.). Three Rbu-P2 carboxylase activase (Rca) polypeptides are encoded in the barley genome by two closely linked, tandemly oriented nuclear genes (RcaA and RcaB); cDNAs encoding each of the three Rbu-P2 carboxylase activase polypeptides were isolated from cDNA libraries of barley leaf mRNA. RcaA produces two mRNAs, which encode polypeptides of 42 and 46 kDa, by an alternative splicing mechanism identical to that previously reported for spinach and Arabidopsis Rca genes (Werneke, J.M., Chatfield, J.M., and Ogren, W. L. (1989) Plant Cell 1, 815-825). RcaB is transcribed to produce a single mRNA, which encodes a mature peptide of 42 kDa. Genomic Southern blots indicate that RcaA and RcaB represent the entire Rbu-P2 carboxylase activase gene family in barley. The genes share 80% nucleotide sequence identity, and the 42-kDa polypeptides encoded by RcaA and RcaB share 87% amino acid sequence identity. Coding regions of the two barley Rca genes are separated by 1 kilobase pair of flanking DNA. DNA sequence motifs similar to those thought to control light-regulated gene expression in other nuclear-encoded plastid polypeptide genes are found at the 5' end of both barley Rca genes. Probes specific to three mRNAs were used to determine the relative contribution each species makes to the total Rca mRNA pool.     

Evidence for a multigene family involved in bile acid 7-dehydroxylation in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is a human intestinal isolate which has an inducible bile acid 7-dehydroxylation activity. At least two cholic acid-induced polypeptides, with molecular masses of 27,000 and 45,000 daltons, respectively, coelute with bile acid 7-dehydroxylation activity. The 45,000-dalton polypeptide appears to be encoded by a cholic acid-induced mRNA species of greater than 6 kilobases, which suggests that the gene coding for this polypeptide is part of a larger operon. A gene has been cloned which flanks the gene encoding the 45,000-dalton polypeptide, in the upstream (5') direction. This gene appears to encode a second 27,000-dalton polypeptide. The gene bears striking homology at both the nucleotide (80%) and deduced amino acid sequence (89%) levels with the gene which encodes the 27,000-dalton polypeptide that has been shown previously to be involved in the bile acid 7-dehydroxylation reaction sequence. The implications of this homology and the possible function(s) of the two homologous genes in bile acid 7-dehydroxylation are discussed. Evidence is presented which suggests that the two homologous genes involved in bile acid 7-dehydroxylation may be part of a larger multigene family in Eubacterium sp. strain VPI 12708.