Nucleotide sequence of a cloned woodchuck hepatitis virus genome: comparison with the hepatitis B virus sequence.

The complete nucleotide sequence of a woodchuck hepatitis virus genome cloned in Escherichia coli was determined by the method of Maxam and Gilbert. This sequence was found to be 3,308 nucleotides long. Potential ATG initiator triplets and nonsense codons were identified and used to locate regions with a substantial coding capacity. A striking similarity was observed between the organization of human hepatitis B virus and woodchuck hepatitis virus. Nucleotide sequences of these open regions in the woodchuck virus were compared with corresponding regions present in hepatitis B virus. This allowed the location of four viral genes on the L strand and indicated the absence of protein coded by the S strand. Evolution rates of the various parts of the genome as well as of the four different proteins coded by hepatitis B virus and woodchuck hepatitis virus were compared. These results indicated that: (i) the core protein has evolved slightly less rapidly than the other proteins; and (ii) when a region of DNA codes for two different proteins, there is less freedom for the DNA to evolve and, moreover, one of the proteins can evolve more rapidly than the other. A hairpin structure, very well conserved in the two genomes, was located in the only region devoid of coding function, suggesting the location of the origin of replication of the viral DNA.     

Role of mutations in the A-subunit of Acholeplasma laidlawii PG-8B topoisomerase IV in formation of resistance to fluoroquinolones

Nucleotide sequence of Acholeplasma laidlawii genome site PG-8B (1000 n.p.), containing topoisomerase IV subunit genes (parE and parC), has been determined. Sequenced genome site contains a gene fragment coding for the C-terminal region of ParE and gene fragment coding for N-terminal region of ParC. Topoisomerase IV subunite genes in A. laidlawii genome are situated near each other and overlapping by 4 nucleotides. Selection in liquid nutrient medium with ascending antibiotic concentrations resulted in derivation of A. laidlawii PG-8B cells resistant to ciprofloxacin, a fluoroquinolone. The resistant clones contain a mutation in the parC QRDR region determining fluoroquinolone resistance: Ser(91) (corresponding to Ser(80) in Escherichia coli ParC) replacement) for Leu.     

Importance of both the coding and the segment-specific noncoding regions of the influenza A virus NS segment for its efficient incorporation into virions

The genome of influenza A virus consists of eight single-strand negative-sense RNA segments, each comprised of a coding region and a noncoding region. The noncoding region of the NS segment is thought to provide the signal for packaging; however, we recently showed that the coding regions located at both ends of the hemagglutinin and neuraminidase segments were important for their incorporation into virions. In an effort to improve our understanding of the mechanism of influenza virus genome packaging, we sought to identify the regions of NS viral RNA (vRNA) that are required for its efficient incorporation into virions. Deletion analysis showed that the first 30 nucleotides of the 3' coding region are critical for efficient NS vRNA incorporation and that deletion of the 3' segment-specific noncoding region drastically reduces NS vRNA incorporation into virions. Furthermore, silent mutations in the first 30 nucleotides of the 3' NS coding region reduced the incorporation efficiency of the NS segment and affected virus replication. These results suggested that segment-specific noncoding regions together with adjacent coding regions (especially at the 3' end) form a structure that is required for efficient influenza A virus vRNA packaging.     

Heterogeneity of Escherichia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes

Phages coding for production of Vero cytotoxins VT1 or VT2 in strains of Escherichia coli serotype O157.H7 or O157.H- were morphologically indistinguishable. Their genome size and restriction enzyme digests of the phage DNA were similar. These phages were clearly different in these respects from a VT1-encoding phage isolated from a strain of E. coli O26.H11 (H19). However the VT1 region cloned from the phage originating in the E. coli O157.H7 strain was identical to the VT1 region previously cloned from the phage carried by H19. Sequences encoding VT2 that were cloned from the phage in E. coli O157.H- have been mapped and the VT2 region identified by transposon insertion. The cloned regions coding for VT1 or VT2 production had no similarities in the presence of restriction enzyme sites over a distance of about 2 kb, and two VT1-specific probes spanning a region of about 1.4 kb did not hybridize under stringent conditions with cloned VT2 DNA. A 2 kb HincII fragment contained the VT2 genes but hybridized to VT1-encoding phages and recombinant plasmids via flanking phage DNA. A 0.85 kb AvaI-PstI fragment was a specific probe for VT2 sequences and did not hybridize under stringent conditions to phages or plasmid recombinants encoding VT1.     

Conserved sequences in both coding and 5'' flanking regions of mammalian opal suppressor tRNA genes.

The rabbit genome encodes an opal suppressor tRNA gene. The coding region is strictly conserved between the rabbit gene and the corresponding gene in the human genome. The rabbit opal suppressor gene contains the consensus sequence in the 3' internal control region but like the human and chicken genes, the rabbit 5' internal control region contains two additional nucleotides. The 5' flanking sequences of the rabbit and the human opal suppressor genes contain extensive regions of homology. A subset of these homologies is also present 5' to the chicken opal suppressor gene. Both the rabbit and the human genomes also encode a pseudogene. That of the rabbit lacks the 3' half of the coding region. Neither pseudogene has homologous regions to the 5' flanking regions of the genes. The presence of 5' homologies flanking only the transcribed genes and not the pseudogenes suggests that these regions may be regulatory control elements specifically involved in the expression of the eukaryotic opal suppressor gene. Moreover the strict conservation of coding sequences indicates functional importance for the opal suppressor tRNA genes.     

Direct immunological identification of full-length cDNA clones for plant protein without gene fusion to E. coli protein

By immunological screening of a cDNA library constructed from potato tuber poly(A)+ RNA and Escherichia coli expression vector pUC8 by the vector-primer and linker procedure of Okayama and Berg [(1982) Mol. Cell Biol. 2, 161-170], nearly full-length cDNA clones for patatin, a major protein of potato tuber, were identified. The cDNA carrying part of the 5'-noncoding region of the patatin mRNA, in addition to entire coding and 3'-noncoding regions, expressed prepatatin in E. coli cells by translational initiation inside cDNA. These results suggest that nearly full-length cDNA clones with entire coding region can be identified directly by immunological screening without gene fusion to E. coli proteins at least for some plant mRNAs.     

Distribution and evolution of sequence characteristics in the E. coli genome

The mean (G + C) composition (51.0%) and standard deviation (+/- 3.8%) of published DNA sequences accounting for 10% of the E. coli genome is in excellent agreement with the principal overall distribution determined by high resolution melting. While differences in base and neighbor characteristics are small and uniform throughout all regions of the genome, it is found that the (G + C) content of sequences varies in segmented fashion within boundaries corresponding to coding (53% G + C) and noncoding (46% G + C) regions; with variances in the latter being six-fold greater than in coding regions. The variance in different regions shows a strong negative dependence on (G + C) content of the region, reflecting the condition that A-T and G-C base pairs are preferred neighbors of A-T and C-G pairs, respectively; with the bias increasing with decreasing (G + C) content. Neighbor analysis indicates the most extreme positive biases occur in AA, TT, GC and CG throughout all regions, but particularly in noncoding regions. Extraordinary numbers of oligomeric strings of (A)n, etc., are the further consequence of this bias. These and other characteristics point to the existence of inherent biases in neighbor frequencies levied during replication or repair, and which reflect, in turn, neighbor influences during mutation. The bias in codon usage noted by Grantham and others is seen here as due, in part, to the adaptation of coding sequences to this microenvironment through selection among synonymous codons so as to preserve inherent neighbor biases.     

A hidden Markov model that finds genes in E. coli DNA.

A hidden Markov model (HMM) has been developed to find protein coding genes in E. coli DNA using E. coli genome DNA sequence from the EcoSeq6 database maintained by Kenn Rudd. This HMM includes states that model the codons and their frequencies in E. coli genes, as well as the patterns found in the intergenic region, including repetitive extragenic palindromic sequences and the Shine-Delgarno motif. To account for potential sequencing errors and or frameshifts in raw genomic DNA sequence, it allows for the (very unlikely) possibility of insertions and deletions of individual nucleotides within a codon. The parameters of the HMM are estimated using approximately one million nucleotides of annotated DNA in EcoSeq6 and the model tested on a disjoint set of contigs containing about 325,000 nucleotides. The HMM finds the exact locations of about 80% of the known E. coli genes, and approximate locations for about 10%. It also finds several potentially new genes, and locates several places were insertion or deletion errors/and or frameshifts may be present in the contigs.     

lacZ gene fusions and insertion mutagenesis in the TL-region of Agrobacterium rhizogenes Ri plasmid

Agrobacterium rhizogenes induces root formation and inserts a fragment of its plasmid into the genome of infected plants. A part of the transferred region (TL-region) of the Ri plasmid of A. rhizogenes strain A4 was cloned in pBR322. Insertions of the Escherichia coli lacZ coding region into the hybrid plasmids were made in vivo using mini-Mu-duction. Two mini-Mus were used, one with the Mu A and B transposase genes (MudII1681) and the other without (MudII1734). Two inserts which result in E. coli lacZ expression where shown to be located in the T-DNA region. This indicates that portions of the T-DNA are capable of expression in bacteria. When these two hybrid plasmids were transformed into Agrobacterium only the one harboring MudII1734 insert gave transformants which correspond to homologous recombination. These results indicate that gene fusion and insertion directed mutagenesis can be simultaneously obtained with this mini-Mu and could be used to study Agrobacterium gene expression.     

Similarities in periodical structures in the position of nucleotides in regions of initiation of replication of bacterial genomes

The regions of initiation of replication of some bacterial genomes were studied by the method of Fourier matrix analysis. A generalized spectral portrait of the primary structures of E. coli-like regions of initiation of replication in bacteria was obtained, which reflects the features of their structural and functional organization. It contains well-pronounced peaks that correspond to the periods T = 2, 11, 17, 27, 86-105 of nucleotides. The peaks corresponding to T = 9, 13, 14, 18, 19, 33-35, 45-47, 74-85, 106-110 are less pronounced. The uniqueness of the Fourier spectrum corresponding to the region of initiation of replication of E. coli oriC was considered by the example of the complete genome of E. coli. Some regions of the E. coli genome were identified that differ from oriC in the primary structure but have Fourier spectra resembling the spectrum of oriC. A number of these regions are alternative points of initiation of replication in sdrA(rnh) mutants of E. coli, the others are localized in yet unidentified regions of the E. coli genome but are capable, in our opinion, to participate in the initiation of replication. Thus, from the similarity of spectral portraits of different regions of the genome, it was possible to reveal several regions that have similar functions, i.e., are involved in initiation of replication.     

A fractal method to distinguish coding and non-coding sequences in a complete genome based on a number sequence representation

A fractal method to distinguish coding and non-coding sequences in a complete genome is proposed, based on different statistical behaviors between these two kinds of sequences. We first propose a number sequence representation of DNA sequences. Multifractal analysis is then performed on the measure representation of the obtained number sequence. The three exponents C(-1), C1 and C2 are selected from the result of multifractal analysis. Each DNA may be represented by a point in the three-dimensional space generated by these three-component vectors. It is shown that points corresponding to coding and non-coding sequences in the complete genome of many prokaryotes are roughly distributed in different regions. Fisher's discriminant algorithm can be used to separate these two regions in the spanned space. If the point (C(-1),C1,C2) for a DNA sequence is situated in the region corresponding to coding sequences, the sequence is discriminated as a coding sequence; otherwise, the sequence is classified as a non-coding one. For all 51 prokaryotes we considered , the average discriminant accuracies pc,pnc,qc and qnc reach 72.28%, 84.65%, 72.53% and 84.18%, respectively.     

大肠杆菌mRNA编码区长度、形成二级结构倾向与密码子偏好性的关系

从GenBank获得大肠杆菌K-12MG1655株的全基因组序列,计算了与基因密码子偏好性相关的多个参数(Nc、CAI、GC、GC3s),对其mRNA编码区长度、形成二级结构倾向与密码子偏好性之间的关系进行了统计学分析,发现虽然翻译效率(包括翻译速度和翻译精度)是制约大肠杆菌高表达基因的密码子偏好性的主要因素,同时,mRNA编码区长度及其形成二级结构的倾向也是形成这种偏好性的不可忽略的原因,而且对偏好性有一定程度的削弱。另外对mRNA编码区形成二级结构倾向的生物学意义进行了讨论分析。     

High-level production of active HIV-1 protease in Escherichia coli.

High levels of active HIV-1 protease (PR) were produced in Escherichia coli, amounting to 8-10% of total cell protein. High production levels were achieved by altering the following parameters: (1) codon preference of the coding region, (2) A+T-richness at the 5' end of the coding region, and (3) promoter. To circumvent the toxicity of HIV-1 PR in E. coli, the gene was expressed as a fusion protein with two different proteolytic autocleavage sequences. In both the cases, the fusion protein could be cleaved in vivo to give an active molecule with the native sequence at the N terminus.