Analysis of the complete nucleotide sequence of the group IV RNA coliphage SP.

We report the nucleotide sequence of the Group IV RNA bacteriophage SP. The entire sequence is 4276 nucleotides long. Four cistrons have been identified by comparison with the related Group III phage Q beta. The maturation protein contains 449 amino acids, the coat protein contains 131 amino acids, the read-through protein contains 330 amino acids and the replicase beta-subunit contains 575 amino acids. SP is 59 nucleotides longer than Q beta. We have analyzed both sequence and structural conservation between SP and Q beta and shown that the sequences for the coat and central region of the replicase are strongly conserved between the two genomes. We also show that the S and M replicase binding sites of Q beta are strongly conserved in SP. Interestingly, the base composition of SP and Q beta differ significantly from one another, and most of the differences can be accounted for by a strong preponderance of U in the third position of each codon of Q beta relative to SP. We also compare conserved hairpins associated with potential coat protein and replicase binding sites.     

Interference with viral infection by RNA replicase deleted at the carboxy-terminal region

Escherichia coli cells harboring an altered Q beta RNA replicase which has amino acid substitutions of the glycine residue at position 357 in the conserved sequence Tyr356-Gly357-Asp358-Asp359 of the beta-subunit protein lost the replicase activity but interfered with proliferation of Q beta phage [Inokuchi and Hirashima (1987) J. Virol. 61, 3946-3949]. To examine the mechanism of the interference, we further analyzed various mutants lacking the carboxy-terminal region of the beta-subunit protein. The cells expressing the beta-subunit gene with up to 17% deletion from the carboxy-terminus of the protein prevented the proliferation of Q beta phage. However, in the case that the deletion extended beyond 25% from the carboxy-terminus, the cells showed no interference. In addition, when the interference took place, the phage coat protein synthesis was inhibited. These results indicate that the region between amino acids 440 and 487 of the beta-subunit protein is involved in the interference and suggest that the defective replicase inhibits the phage coat protein synthesis by competing with the ribosomes at the initiation site of the coat gene.     

Characterization of an intergroup serological mutant from group II RNA phage GA

Starting from the group II RNA phage GA which has an amber mutation in the maturation protein cistron, a spontaneous mutant of group II phage GA, whose serological and electrophoretic properties became similar to those of group I phage MS2, was isolated and analyzed. The mutant has now become sensitive to anti-MS2 serum and resistant to anti-GA serum. Analysis of the nucleotide sequence of the coat protein gene revealed that G----A transition was the main change. The deduced amino acid sequence showed that five amino acids were substituted in the mutant, and three of the five became identical to MS2, resulting in increased molecular weight of the coat protein. However, it did not complement MS2. These results suggested that the serological change from group II phage GA type to group I phage MS2 type is induced spontaneously at high frequency by minor nucleotide changes in coat protein gene, and confirmed the previous results at the RNA level that MS2 and GA were related although the closeness between them seems somewhat remoter than that of groups III and IV (18, Inokuchi et al, unpublished data for the nucleotide sequence of group IV phage SP).     

Molecular cloning and the complete nucleotide sequence of the creatine kinase-M cDNA from chicken.

The nucleotide sequence of creatine kinase-M (CK-M) cDNA clones has been determined. It includes the entire coding region of 381 amino acids in addition to 5' and 3' untranslated regions. A comparison with a partial sequence from rat CK-M reveals 84% nucleotide sequence homology in the coding region but divergence in the 3' untranslated region. The amino acid sequence is 94% conserved between chicken and rat. Hybridization to RNA immobilized on filters indicates homology between the CK-M 3' untranslated region and additional muscle specific RNA species. The coding region hybridizes only to CK-M RNA.     

Genome structure of caulobacter phage phiCb5

The complete genome sequence of caulobacter phage phiCb5 has been determined, and four open reading frames (ORFs) have been identified and characterized. As for related phages, the ORFs code for maturation, coat, replicase, and lysis proteins, but unlike other Leviviridae members, the lysis protein gene of phiCb5 entirely overlaps with the replicase in a different reading frame. The lysis protein of phiCb5 is about two times longer than that of the distantly related MS2 phage and presumably contains two transmembrane helices. Analysis of the proposed genome secondary structure revealed a stable 5' stem-loop, similar to other phages, and a substantially shorter 3' untranslated region (UTR) structure with only three stem-loops.     

RNA binding properties of the coat protein from bacteriophage GA.

The coat protein of bacteriophage GA, a group II RNA phage, binds to a small RNA hairpin corresponding to its replicase operator. Binding is specific, with a Ka of 71 microM -1. This interaction differs kinetically from the analogous coat protein-RNA hairpin interactions of other RNA phage and also deviates somewhat in its pH and salt dependence. Despite 46 of 129 amino acid differences between the GA and group I phage R17 coat proteins, the binding sites are fairly similar. The essential features of the GA coat protein binding site are a based-paired stem with an unpaired purine and a four nucleotide loop having an A at position -4 and a purine at -7. Unlike the group I phage proteins, the GA coat protein does not distinguish between two alternate positions for the unpaired purine and does not show high specificity for a pyrimidine at position -5 of the loop.     

鲑鱼生长激素cDNA的分子克隆和序列分析

从太平洋切奴克鲑鱼(Pacific Chinook Salmon,Oncorthychus tschawytscha)垂体poly(A)~+ RNA构建cDNA文库。按照鲑鱼生长激素(sGH)部分氨基酸序列合成两个寡聚脱氧核苷酸探针,它们分别与编码第1—7和第166—172氨基酸序列互补。用探针筛查cDNA文库,得到了完整的sGH cDNA克隆。cDNA序列已测定,包括编码210个氨基酸的编码序列。其中含有22个氨基酸的信号肽序列和188个氨基酸的成熟GH序列。该克隆还包括了5'端和3'端非翻译区,分别为72个和438个碱基对长。与Chum鲑鱼比较表明,核酸序列和氨基酸序列的同源性分别为97%和99%。     

Complete nucleotide sequence of genomic RNA of the potato M-virus

The complete nucleotide sequence of potato virus M genomic RNA has been determined to be 8534 nucleotides (with the exception of the poly(A) tail at the 3' end). The sequence contains six large open reading frames coding for proteins of mol. wt. 223206, 25438, 11893, 6793, 33906, and 12183 (in 5'----3' direction). According to its primary sequence analysis the 223K protein ORF codes for a virus RNA replicase. The in vitro translation product of 34K protein gene precipitates by the antisera against the RVM indicating that the 34K protein is the virus coat protein. The general aspects of carla- and potexvirus gene organization are discussed.     

Complete nucleotide sequence of the group I RNA bacteriophage fr

We report the complete nucleotide sequence of the group I RNA bacteriophage fr. The entire genome consists of 3575 nucleotides, six nucleotides more than the only other sequenced group I representative, MS2. The greatest divergence between these phages occurs in the 5' terminal region of the A gene, while the lysis-replicase gene overlap, the coat gene and the central region of the replicase gene are highly conserved. Overall sequence homology between fr and MS2 is 77%. Here, we present a general comparison between the two phages. In the accompanying paper we use phylogenetic sequence comparison between MS2 and fr to deduce the secondary structure at the 3' untranslated region.     

Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing

We have isolated and characterized two distinct myosin heavy chain cDNA clones from a neonatal rat aorta cDNA library. These clones encode part of the light meromyosin region and the carboxyl terminus of smooth muscle myosin heavy chain. The two rat aorta cDNA clones were identical in their 5' coding sequence but diverged at the 3' coding and in a portion of the 3' untranslated regions. One cDNA clone, RAMHC21, encoded 43 unique amino acids from the point of divergence of the two cDNAs. The second cDNA clone, RAMHC 15, encoded a shorter carboxyl terminus of nine unique amino acids and was the result of a 39 nucleotide insertion. This extra nucleotide sequence was not present in RAMHC21. The rest of the 3' untranslated sequences were common to both cDNA clones. Genomic cloning and DNA sequence analysis demonstrated that an exon specifying the 39 nucleotides unique to RAMHC15 mRNA was present, together with the 5' upstream common exons in the same contiguous stretch of genomic DNA. The 39 nucleotide exon is flanked on either side by two relatively large introns of approximately 2600 and 2700 bases in size. RNase protection analysis indicated that the two corresponding mRNAs were coexpressed in both vascular and non-vascular smooth muscle tissues. This is the first demonstration of alternative RNA processing in a vertebrate myosin heavy chain gene and provides a novel mechanism for generating myosin heavy chain protein diversity in smooth muscle tissues.     

The lysis function of RNA bacteriophage Qbeta is mediated by the maturation (A2) protein

Complete or partial cDNA sequences of the RNA bacteriophage Qbeta were cloned in plasmids under the control of the lambdaP(L) promoter to allow regulated expression in Escherichia coli harbouring the gene for the temperature-sensitive lambdaCI857 repressor. Induction of the complete Qbeta sequence leads to a 100-fold increase in phage production, accompanied by cell lysis. Induction of the 5'-terminal sequence containing the intact maturation protein (A2) cistron also causes cell lysis. Alterations of the A2 cistron, leading to proteins either devoid of approximately 20% of the C-terminal region or of six internal amino acids, abolish the lysis function. Expression of other cistrons in addition to the A2 cistron does not enhance host lysis. Thus, in Qbeta, the A2 protein, in addition to its functions as maturation protein, appears to trigger cell lysis. This contrasts with the situation in the distantly related group I RNA phages such as f2 and MS2 where a small lysis polypeptide is coded for by a region overlapping the end of the coat gene and the beginning of the replicase gene.     

The complete nucleotide sequence of murine beta-glucuronidase mRNA and its deduced polypeptide

The complete nucleotide sequence of murine beta-glucuronidase (GUS) mRNA has been compiled from three overlapping cloned cDNAs and a single GUS-specific genomic clone. The sequence is composed of 2455 nucleotides, exclusive of the poly(A) tail. The 5' and 3' untranslated regions contain 12 and 499 bases, respectively, with the open reading frame encoding a polypeptide of 648 amino acids (74.2 kDa), including a 22 amino acid signal sequence. The nucleotide and deduced amino acid sequences of murine GUS are compared to those published for rat and human GUS and the results are presented. Murine GUS also shares amino acid sequence identity with Escherichia coli GUS and beta-galactosidase. The complete sequences of murine GUS mRNA and its deduced polypeptide provide a basis from which to study the mechanisms responsible for the well-characterized variation in GUS expression among inbred mouse strains.     

In vitro genetic selection analysis of alfalfa mosaic virus coat protein binding to 3'-terminal AUGC repeats in the viral RNAs.

The coat proteins of alfalfa mosaic virus (AMV) and the related ilarviruses bind specifically to the 3' untranslated regions of the viral RNAs, which contain conserved repeats of the tetranucleotide sequence AUGC. The purpose of this study was to develop a more detailed understanding of RNA sequence and/or structural determinants required for coat protein binding by characterizing the role of the AUGC repeats. Starting with a complex pool of 39-nucleotide RNA molecules containing random substitutions in the AUGC repeats, in vitro genetic selection was used to identify RNAs that bound coat protein. After six iterative rounds of selection, amplification, and reselection, 25% of the RNAs selected from the randomized pool were wild type; that is, they contained all four AUGC sequences. Among the 31 clones analyzed, AUGC was clearly the preferred selected sequence at the four repeats, but some nucleotide sequence variability was observed at AUGC(865-868) if the other three AUGC repeats were present. Variant RNAs that bound coat protein with affinities equal to or greater than that of the wild-type molecule were not selected. To extend the in vitro selection results, RNAs containing specific nucleotide substitutions were transcribed in vitro and tested in coat protein and peptide binding assays. The data strongly suggest that the AUGC repeats provide sequence-specific determinants and contribute to a structural platform for specific coat protein binding. Coat protein may function in maintaining the 3' ends of the genomic RNAs during replication by stabilizing an RNA structure that defines the 3' terminus as the initiation site for minus-strand synthesis.     

Partial amino acid sequence of human thrombospondin as determined by analysis of cDNA clones: homology to malarial circumsporozoite proteins

A lambda gt 11 library prepared from human umbilical vein endothelial cell RNA was screened for cDNAs encoding thrombospondin. Reagents included a monospecific antibody to human thrombospondin and a mixture of four synthetic oligodeoxyribonucleotides derived from an amino acid sequence near the NH2 terminus of mature human thrombospondin. Two series of cDNA clones coding for sequences at the 5' and 3' ends of thrombospondin mRNA, respectively, were isolated. The nucleotide sequence of a 1.3-kilobase (kb) 5' clone (lambda TS-33) coded for 99 bases of 5' untranslated RNA, a signal peptide of 18 amino acids, and the first 379 amino acids of thrombospondin. Northern blot analysis with lambda TS-33 detected a single mRNA species of approximately 6.0 kb in rat aortic smooth muscle cell RNA. Thrombospondin mRNA levels increased rapidly, but transiently, in quiescent smooth muscle cells treated with platelet-derived growth factor. The kinetics of this response were very similar to those of the thrombospondin protein to this growth factor. There was significant homology in amino acid sequence between thrombospondin and a conserved region in the circumsporozoite protein of two malarial sporozoites. This region of thrombospondin may therefore represent a potential recognition site for a cell surface thrombospondin receptor.     

Nucleotide sequence and structural organization of the 3'-terminal region of potato virus M

The sequence of 2630 3'-terminal nucleotides has been determined for the genomic RNA of potato virus M (PVM), a type member of the carlavirus group. Analysis of this nucleotide sequence revealed five open reading frames coding for proteins of mol. wt. 25, 12, 7, 34 and 11 kDa (in 5'----3' direction). The PVM genome organization has been shown to be basically analogous to that of potexviruses, except that the latter at their 3' end lack the gene encoding 11K protein. Amino acid sequence comparison between the PVM 34K protein and the coat proteins of potexviruses has shown a high extent of homology in the C-terminal amino acids. Homology has also been found between the 25, 12 and 7K proteins encoded by PVM RNA and corresponding potexvirus proteins. All this gave grounds for suggestion that carlaviruses and potexviruses belong to the same subgroup of phytoviruses.     

Sequencing and analysis for the full-length genome RNA of foot-and-mouth disease virus China/99

The complete nucleotide sequence of genomic RNA of foot and mouth disease virus (FMDV) strain China/99 from infected bovine tongue epithelium is presented. The nucleotide sequence extending from the 5' end of the genomic RNA to the 5' end of poly (A) tail contains 8173 nucleotides (nt). Its open reading frame, which encodes a single polypeptide of 2332 amino acids, encompasses 6999 nt starting from the initiation codon AUG and terminating at the UAA codon 93 bases upstream from the 5' end of poly (A) tract. The 5' untranslated region (UTR) is composed of 1081 nt. The consensus of the 1d gene of FMDV strain China/99 compared with that of UKG/6/2001, UKG/12/2001, China/99HN4 and China/3/Tibet is over 97%. The result showed the stains belong to the members of the Pan-Asia family. There is a remarkable differentiation in the function-unknown (FUR), p2 and p3 regions between FMDV isolates from infected cattle and swine, especially in 3a gene. No deletion was found in genes /, 1a, 1b, 2a, 2c, 3b, and 3d. Thes     

Primary and secondary structure of black beetle virus RNA2, the genomic messenger for BBV coat protein precursor.

The nucleotide sequence of black beetle virion (BBV) RNA2 has been determined. RNA2 is 1399 b long. Its 5' terminus is capped. Its 3' terminus has an unidentified moiety that renders the RNA resistent to polyadenylation and ligation. The first AUG codon at base 23 is followed by an open reading frame for a protein 407 amino acids long, the predicted size of coat protein precursor. A second open reading frame for a putative protein 72 amino acid residues long begins at base 1110. No other large open reading frames exist. The 5' half of the RNA can be folded into a long, imperfect hairpin of high predicted stability. The 3' half of the RNA can fold into a complex set of multiply bifurcated stem and loop regions.