Nucleotide sequence of an infectious molecularly cloned genome of ground squirrel hepatitis virus.

We have determined the complete nucleotide sequence of an infectious cloned genome of ground squirrel hepatitis virus (GSHV), a nonpathogenic member of the hepadnavirus group. The genome is 3,311 base pairs long and contains the major open reading frames described for the related human and woodchuck hepatitis B viruses (HBV and WHV, respectively). These reading frames include genes for the major structural proteins (the surface and core antigens), unassigned open reading frames (A and B), the longer of which is presumed to encode the viral DNA polymerase, and an open reading frame preceding and continuous with the surface antigen gene. The arrangement of these open reading frames is similar to that encountered in the genomes of HBV and WHV: all of the reading frames are encoded on the same strand, they are positioned in the same fashion with respect to each other, and a large portion (at least 51%) of the genome can be translated in two reading frames. Comparisons of the predicted translational products of the three mammalian hepadnaviruses reveal 78% amino acid homology between the proteins of GSHV and WHV and 43% homology between those of GSHV and HBV. In addition, a perfect direct repeat of 10 to 11 base pairs, separated by ca. 46 to 223 base pairs, is present in the three mammalian viruses and in duck hepatitis B virus; the position of the repeats near the 5' termini of the two strands of virion DNA suggests a role in viral replication.     

Predicting Statistical Properties of Open Reading Frames in Bacterial Genomes

An analytical model based on the statistical properties of Open Reading Frames (ORFs) of eubacterial genomes such as codon composition and sequence length of all reading frames was developed. This new model predicts the average length, maximum length as well as the length distribution of the ORFs of 70 species with GC contents varying between 21% and 74%. Furthermore, the number of annotated genes is predicted with high accordance. However, the ORF length distribution in the five alternative reading frames shows interesting deviations from the predicted distribution. In particular, long ORFs appear more often than expected statistically. The unexpected depletion of stop codons in these alternative open reading frames cannot completely be explained by a biased codon usage in the +1 frame. While it is unknown if the stop codon depletion has a biological function, it could be due to a protein coding capacity of alternative ORFs exerting a selection pressure which prevents the fixation of stop codon mutations. The comparison of the analytical model with bacterial genomes, therefore, leads to a hypothesis suggesting novel gene candidates which can now be investigated in subsequent wet lab experiments.     

Expression and functional analysis of glutamate synthase small subunit-like proteins from archaeon Pyrococcus horikoshii

Glutamate synthase, glutamine α-ketoglutarate amidotransferase (often abbreviated as GOGAT) is a key enzyme in the early stages of ammonia assimilation in bacteria, algae and plants, catalyzing the reductive transamidation of the amido nitrogen from glutamine to α-ketoglutarate to form two molecules of glutamate. Most bacterial glutamate synthases consist of a large and small subunit. The genomes of three Pyrococcus species harbour several open reading frames which show homology with the small subunit of glutamate synthase. There are no open reading frames which may be coding for a large subunit responsible for the glutamate formation in these pyrococcal genomes.In this work, two open reading frames PH0876 and PH1873 from P. horikoshii were cloned and expressed in Escherichia coli as soluble proteins. Both proteins show NADPH-dependent oxidoreductase activity using artificial electron acceptors iodonitrotetrazolium chloride at thermophilic conditions. It is possible that these open reading frames are the products of gene duplication and that they are the early forms of an electron transfer domain in archaea which may have later contributed to many electron transfer enzymes.     

Many human endogenous retrovirus K (HERV-K) proviruses are unique to humans.

BACKGROUND: Endogenous retroviruses contribute to the evolution of the host genome and can be associated with disease. Human endogenous retrovirus K (HERV-K) is related to the mouse mammary tumor virus and is present in the genomes of humans, apes and cercopithecoids (Old World monkeys). It is unknown how long ago in primate evolution the full-length HERV-K proviruses that are in the human genome today were formed. RESULTS: Ten full-length HERV-K proviruses were cloned from the human genome. Using provirus-specific probes, eight of the ten were found to be present in a genetically diverse set of humans but not in other extant hominoids. Intact preintegration sites for each of these eight proviruses were present in the apes. A ninth provirus was detected in the human, chimpanzee, bonobo and gorilla genomes, but not in the orang-utan genome. The tenth was found only in humans, chimpanzees and bonobos. Complete sequencing of six of the human-specific proviruses showed that full-length open reading frames for the retroviral protein precursors Gag-Pro-Pol or Env were each present in multiple proviruses. CONCLUSIONS: At least eight full-length HERV-K genomes that are in the human germline today integrated after humans diverged from chimpanzees. All of the viral open reading frames and cis-acting sequences necessary for HERV-K replication must have been intact during the recent time when these proviruses formed. Multiple full-length open reading frames for all HERV-K proteins are present in the human genome today.     

Nucleotide sequence of a cloned woodchuck hepatitis virus genome: evolutional relationship between hepadnaviruses.

We have determined the complete nucleotide sequence of a cloned DNA of woodchuck hepatitis virus (WHV), the most oncogenic virus among hepadnaviruses. The genome, designated WHV2, is 3,320 base pairs long and contains four major open reading frames (ORFs) coded on the same strand of nucleotide sequence as in the human hepatitis B virus (HBV) genome. Comparison of the nucleotide sequence and amino acid sequences deduced from it among the genomes of various hepadnaviruses demonstrates that each protein shows an intrinsic property in conserving its amino acid sequence. A parameter, the ratio of the number of triplets with one-letter change but no amino acid substitution to the total number of triplets in which one-letter change occurred, was introduced to measure the intrinsic properties quantitatively. For each ORF, the parameter gave characteristic values in all combinations. Therefore, the relative evolutional distance between these hepadnaviruses can be measured by the amino acid substitution rate of any ORF. These comparisons suggest that (i) the difference between two WHV clones, WHV1 and WHV2, corresponds to that among clones of a HBV subtype, HBVadr, and (ii) WHV and ground squirrel hepatitis virus can be categorized in a way similar to the subgroups of HBV.     

An agent-based system for re-annotation of genomes

Genome annotation projects can produce incorrect results if they are based on obsolete data or inappropriate models. We have developed an automatic re-annotation system that uses agents to perform repetitive tasks and reports the results to the user. These tasks involve BLAST searches on biological databases (GenBank) and the use of detection tools (Genemark and Glimmer) to identify new open reading frames. Several agents execute these tools and combine their results to produce a list of open reading frames that is sent back to the user. Our goal was to reduce the manual work, executing most tasks automatically by computational tools. A prototype was implemented and validated using Mycoplasma pneumoniae and Haemophilus influenzae original annotated genomes. The results reported by the system identify most of new features present in the re-annotated versions of these genomes.     

微生物小开放阅读框:小蛋白及翻译调控研究进展

小开放阅读框(small open reading frame, sORF)广泛存在于不同生物基因组中,由于其序列短,以及编码的产物小蛋白(smallprotein,或称微蛋白;microprotein或迷你蛋白miniprotein)检测困难等原因,小开放阅读框长期未得到充分注释和研究。近年来,随着高通量测序、翻译组和质谱分析等技术的不断发展,在不同生物中发现大量新的小开放阅读框,其编码的小蛋白及介导的翻译调控已应用于药物开发及植物抗病机理等研究。但是,目前对微生物的小开放阅读框相关研究和应用还相对有限。本文综述了小开放阅读框编码产物小蛋白的发现和鉴定,以及上游开放阅读框(upstream open reading frame, uORF)对mRNA翻译调控等最新研究进展,重点介绍了微生物基因组中小开放阅读框的鉴定和功能研究进展,为深入认识微生物中小开放阅读框的功能和作用机制,以及植物和动物等高等其他生物的小蛋白和翻译调控相关研究提供参考。     

Distinguishing the ORFs from the ELFs: short bacterial genes and the annotation of genomes

A substantial fraction of hypothetical open reading frames (ORFs) in completely sequenced bacterial genomes are short, suggesting that many are not genes but random stretches of DNA. Although it is not feasible to authenticate the coding capacity of all such regions experimentally, comparisons of ORFs in related genomes can expose those that encode functional proteins.     

5’UTR中AUG的分布及其对翻译起始的影响

以细菌和古菌基因组5′ UTR序列作为研究对象,分析在5′ UTR 的3个不同阅读框架中三联体AUG的分布,发现无论是细菌还是古菌基因组都在阅读框1中有非常明显的AUG缺失(depletion)。AUG的缺失表明在起始密码子上游的AUG很可能会对基因的翻译起始产生影响。分析得知：绝大部分的AUG都是以uORF(upstream open reading frame)的形式出现的,uAUG(upstream AUG)的数量很少,特别是在阅读框1中,而且在细菌基因组的阅读框1中uAUG较多地出现在了含有SD序列的基因上游。比较发现,uAUG引导的序列在同义密码子使用上的偏好性较真正的编码序列差,这可能表明细菌和古菌在同义密码子使用上的偏好性也是决定基因准确地翻译起始的重要因素之一。     

Organization and variation of angiosperm mitochondrial genome

The mitochondrial genomes of angiosperms are the largest mitochondrial genomes so far reported and are highly variable in size among plant species. The comparative analysis of the angiosperm mitochondrial genomes at the nucleotide level has now become feasible for addressing long-standing questions, owing to the publication of five dicot and three monocot genomes. Whereas the identified genes and introns are rather well conserved, intergenic regions are highly variable in sequence, even between two close relatives. Promiscuous DNA and horizontally transferred sequence constitute part of the intergenic regions, but the origin of the majority of these regions is unknown. On the other hand, duplication and extensive rearrangement of preexisting sequences may be one of the explanations for the occurrence of unknown sequences. Functional aspects of the mitochondrial genome, such as RNA editing and expression of unique open reading frames (ORFs), can be changed under certain nuclear genotypes.     

Integration of human papillomavirus type 16 DNA sequences: a possible early event in the progression of genital tumors.

The keratinocyte line SK-v harbors only integrated human papillomavirus type 16 (HPV 16) DNA sequences, although it originated from vulvar Bowenoid papules predominantly containing multiple copies of free HPV 16 genomes. We have cloned a fragment of cell DNA that contains the integrated HPV 16 DNA sequences and have shown that integration interrupts the HPV 16 genome in open reading frames E2 and L2 and creates a deletion of 813 base pairs. This allows the expression of open reading frames E6 and E7, as actually substantiated by Northern (RNA) blot analysis of SK-v RNAs with subgenomic HPV 16 RNA probes. Using a unique flanking cellular DNA sequence as the probe, we have shown that the integration of HPV 16 sequences had already occurred in the premalignant lesions from which the SK-v cell line was derived.     

Detecting overlapping coding sequences with pairwise alignments

MOTIVATION: Overlapping gene coding sequences (CDSs) are particularly common in viruses but also occur in more complex genomes. Detecting such genes with conventional gene-finding algorithms can be difficult for several reasons. If an overlapping CDS is on the same read-strand as a known CDS, then there may not be a distinct promoter or mRNA. Furthermore, the constraints imposed by double-coding can result in atypical codon biases. However, these same constraints lead to particular mutation patterns that may be detectable in sequence alignments. RESULTS: In this paper, we investigate several statistics for detecting double-coding sequences with pairwise alignments--including a new maximum-likelihood method. We also develop a model for double-coding sequence evolution. Using simulated sequences generated with the model, we characterize the distribution of each statistic as a function of sequence composition, length, divergence time and double-coding frame. Using these results, we develop several algorithms for detecting overlapping CDSs. The algorithms were tested on known overlapping CDSs and other overlapping open reading frames (ORFs) in the hepatitis B virus (HBV), Escherichia coli and Salmonella typhimurium genomes. The algorithms should prove useful for detecting novel overlapping genes--especially short coding ORFs in viruses. AVAILABILITY: Programs may be obtained from the authors. SUPPLEMENTARY INFORMATION: http://biochem.otago.ac.nz/double.html.     

Comparative analysis of the human type 1a and bovine type 1 papillomavirus genomes.

The DNA sequences of the genomes of the bovine type 1 and human type 1a papillomaviruses were compared. The overall organization of both genomes is very similar. Three areas of maximal homology were found in the L1 and E1/E2 genes, and at the beginning of L2. The conservation of homologous amino acid sequences encoded in the open reading frames argues that these segments represent real genes or exons. Within these segments, however, only certain domains of the putative proteins are preferentially conserved. Two polypeptide chains show homologous arrangement of the cysteine residue clusters Cys-X-X-Cys, despite a lack of conservation of the rest of the amino acid sequence. A significant sequence divergence in a region where the three reading frames are open suggests that papillomavirus genomes have evolved not solely by accumulation of point mutations. Conserved sequences were also found in the noncoding region, and their possible involvement in regulation of viral gene expression is discussed.     

植物小开放阅读框编码肽的研究进展

小开放阅读框（small open reading frame,sORF）一般指基因组中能够编码长度在100个氨基酸左右或以内短肽的开放阅读框。它们广泛存在于植物基因组,却因编码短肽而常被基因组注释忽视。随着翻译组学和蛋白质组学测序技术的发展,具有翻译活性的sORF被证实广泛存在于植物基因组,且参与植物生长发育等重要过程的调控。该文归纳了近些年来植物领域sORF的一些研究进展,主要包括sORF的来源与分类、信息学预测方法和生物学功能等,并基于此对植物sORF未来的研究方向进行了展望。     

Characterization of a hypervariable region in the genome of Chlamydophila pneumoniae

Chlamydophila pneumoniae displays surprisingly little genomic variation, as seen by comparisons of the published genomes from three different isolates and sequencing of four different genes from different isolates. We have in the present study, however, demonstrated genomic variation between 10 C. pneumoniae isolates in the 11690-bp region between the two outer membrane protein genes pmp1 and pmp2. This region of the C. pneumoniae CWL-029 isolate contains seven C. pneumoniae-specific open reading frames (hb1-7, encoding hydrophobic beta-sheet-containing proteins). We identified additionally 12 open reading frames in the C. pneumoniae CWL-029 genome encoding hypothetical proteins with similarity to the seven hypothetical Hb-proteins. Compared to other isolates, genomic variation is seen to cause frame-shifting of three of the 19 hb-open reading frames, which are proposed to be three full-length genes and eight frame-shifted pseudogenes. The hypothetical proteins encoded by these proposed genes contain an N-terminally located highly hydrophobic stretch of 50-60 residues. A similar motif is found in all identified Chlamydia inclusion membrane proteins and therefore the Hb-proteins are candidate inclusion proteins.     

Small mobile sequences in bacteria display diverse structure/function motifs

Small repeat sequences in bacterial genomes, which represent non-autonomous mobile elements, have close similarities to archaeon and eukaryotic miniature inverted repeat transposable elements. These repeat elements are found in both intergenic and intragenic chromosomal regions, and contain an array of diverse motifs. These can include DNA sequences containing an integration host factor binding site and a proposed DNA methyltransferase recognition site, transcribed RNA secondary structural motifs, which are involved in mRNA regulation, and translated open reading frames found fused to other open reading frames. Some bacterial mobile element fusions are in evolutionarily conserved protein and RNA genes. Others might represent or lead to creation of new protein genes. Here we review the remarkable properties of these small bacterial mobile elements in the context of possible beneficial roles resulting from random insertions into the genome.     

Conservation of amino acid transporters in fungi, plants and animals

When comparing the transporters of three completely sequenced eukaryotic genomes--Saccharomyces cerevisiae, Arabidopsis thaliana and Homo sapiens--transporter types can be distinguished according to phylogeny, substrate spectrum, transport mechanism and cell specificity. The known amino acid transporters belong to five different superfamilies. Two preferentially Na(+)-coupled transporter superfamilies are not represented in the yeast and Arabidopsis genomes, whereas the other three groups, which often function as H(+)-coupled systems, have members in all investigated genomes. Additional superfamilies exist for organellar transport, including mitochondrial and plastidic carriers. When used in combination with phylogenetic analyses, functional comparison might aid our prediction of physiological functions for related but uncharacterized open reading frames.