Genetic definition of a new bovine papillomavirus type 1 open reading frame, E5B, that encodes a hydrophobic protein involved in altering host-cell protein processing.

We have previously observed that bovine papillomavirus type 1 (BPV-1) induces the appearance of five cellular proteins in C127 mouse fibroblasts, four of which appear to arise by altered processing of resident endoplasmic reticulum proteins. Studies of various cell lines revealed that expression of the 3' end of the BPV early region was sufficient for induction of these changes. To identify the BPV gene responsible, we have utilized the simian virus 40 (SV40)/BPV-1 recombinant virus Pava-1, which expresses the 3' end of the BPV early region behind an SV40 early promoter. C127 cells infected with Pava-1 for 48 h show the expected BPV-associated alterations, as do cells infected with Pava constructs mutated in the E5 or E2 genes. However, a mutation in the start codon of a previously ignored open reading frame extending from nucleotides 4013 to 4170 (E5B) eliminated the BPV-associated changes. Similar results were obtained with COS cells infected with the Pava mutants and C127 cells transformed by full-length mutated BPV. Despite its influence on the processing of cellular endoplasmic reticulum proteins, this mutation in E5B did not alter BPV-transforming efficiency or the ability of transformants to form colonies in soft agar. The E5B open reading frame encodes a hydrophobic 52-amino-acid polypeptide that shares structural similarities with HPV6 E5A and HPV16 E5. Speculations on a role for E5B in the viral life cycle are discussed.     

Identification of a 68-kilodalton nuclear ATP-binding phosphoprotein encoded by bovine papillomavirus type 1.

E1 is the largest open reading frame (ORF) of bovine papillomavirus type 1 (BPV-1) and is highly conserved among all papillomaviruses, maintaining its size, amino acid composition, and location in the viral genome with respect to other early genes. Multiple viral replication functions have been mapped to the E1 ORF of BPV-1, and evidence suggested that more than one protein was encoded by this ORF. We previously identified a small protein (M) whose gene consists of two exons, one encoded by the 5' end of the E1 ORF. We show here that a 68-kilodalton (kDa) phosphoprotein made from the E1 ORF can be detected in BPV-1-transformed cells, and we present evidence that this protein is encoded by sequences colinear with the entire E1 ORF. The full-length E1 protein immunoprecipitated from virally transformed cells and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis comigrates with a protein expressed from a recombinant DNA construct capable of producing only the complete E1 protein. In addition, two different antisera directed against polypeptides encoded from either the 3' or the 5' end of the E1 ORF both recognize the full-length E1 product. A mutation converting the first methionine codon in the ORF to an isoleucine codon abolishes BPV-1 plasmid replication and E1 protein production. Consistent with the notion that this methionine codon is the start site for E1, a mutant with a termination codon placed after the splice donor at nucleotide 1235 in E1 produces a truncated protein with the molecular mass predicted from the primary sequence as well as the previously identified M protein. When visualized by immunostaining, the E1 protein expressed in COS cells is localized to the cell nucleus. A high degree of similarity exists between the BPV-1 E1 protein and polyomavirus and simian virus 40 large-T antigens in regions of the T antigens that bind ATP. We show by ATP affinity labeling that the E1 protein produced in COS cells binds ATP and that this activity is abolished by a point mutation which converts the codon for proline 434 to serine. Furthermore, this mutation renders the viral genome defective for DNA replication, suggesting that the ATP-binding activity of E1 is necessary for its putative role in viral DNA replication.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular cloning and nucleotide sequence of deer papillomavirus.

The genome of deer papillomavirus (DPV) isolated from American white-tailed deer was cloned into pBR322, and the entire nucleotide sequence of 8,374 base pairs was determined. The overall genetic organization of the DPV genome was similar to that of other papillomaviruses. All significant open reading frames were located on one strand, and the locations of putative promoters and polyadenylation signals were similar to those identified in the closely related bovine papillomavirus type 1 (BPV-1) genome. The DPV genome was approximately colinear with BPV-1 except for a noncoding region separating the early and late regions. The regions of highest nucleotide sequence homology between DPV and BPV-1 were found in the E1 open reading frame coding for BPV-1 DNA replication function and in the L1 open reading frame, which encodes the major capsid protein of BPV-1.     

一种与抗生素调控基因afsk高度同源的新基因——Spy1的克隆

普那霉素是由始旋链霉菌产生的一种链阳性菌素 类抗生素.目前国外对普那霉素的基因工程研究甚少,国内尚属空白,这阻碍了利用基因工程 的方法来提高普那霉素产量的发展.本文通过对普那霉素产生菌——始旋链霉菌柯斯基因组 文库的构建和菌落原位杂交,筛选出了与普那霉素高产密切相关的新基因所在的柯斯质粒,并通过 酶切分析和Southern杂交确定了该基因所在的酶切片段,对酶切片段进行亚克隆测序,获得了 与天蓝色链霉菌中抗生素调控基因afsk同源新基因的全长克隆,该新基因包含有1个2 127 bp的开放阅读框(ORF),编码708个氨基酸的蛋白,命名为Spy1.对该新基因的生物信息学初步分析表明,该基因编码的是丝/苏氨酸蛋白激酶.