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H Shimojo

Publications and source records attributed to H Shimojo.

At least 37 records · Page 2Linked to original sources
PubMed1984

Characterization of a host range mutant of human adenovirus 12 defective in early region 1B.

We isolated an adenovirus 12 early region 1B mutant (in206B) by ligation of the cleaved DNA-protein complex and transfection of human embryo kidney cells with the ligation products. By deduction from the DNA sequencing analysis, the two polypeptides (with molecular weights of 19,000 and 54,000) coded in the early region 1B were fused in this mutant to produce a large polypeptide. This mutant could replicate efficiently in 293 cells but not efficiently in KB or human embryo kidney cells. In KB cells, viral DNA replication could not be detected after infection with in206B. In human embryo kidney cells, viral DNA replication did occur, but the transition of viral mRNA patterns from early to late did not occur even after DNA replication, resulting in failure to produce the late polypeptides. These results indicate that the early region 1B products may be involved in both viral DNA replication and regulation of transcription.

Adenoviruses, Human↗
PubMed1984

Expression of the E4 gene is required for establishment of soft-agar colony-forming rat cell lines transformed by the adenovirus 12 E1 gene.

Rat 3Y1 cells were transfected with recombinant gARC ( pSV2gpt carrying the adenovirus 12 early region 1 [E1] gene), and focus formation was observed in monolayer cultures after culture of cells in gpt-selective medium (Eagle medium containing 10% fetal calf serum, xanthine, thymidine, aminopterin, and mycophenolic acid) for 10 days, followed by focus formation. Transformed E1Y cell lines were then established from these foci. The E1Y cells were transformed morphologically similarly to cells transformed with intact adenovirus 12 DNA but formed no colonies in soft-agar culture and induced tumors in transplanted rats only after a long incubation period. For the establishment of completely transformed cells, 3Y1 cells were transformed with combinations of gARC , pE3 (pBR322 carrying the adenovirus 12 E3 gene), and gE4 ( pSV2gpt carrying the adenovirus 12 E4 gene) DNA. E1- 3Y cells (3Y1 cells transformed with gARC and pE3 DNA), E1- 4Y cells (3Y1 cells transformed with gARC and gE4 DNA), and E1-3- 4Y cells (3Y1 cells transformed with gARC , pE3 , and gE4 DNA) were established. These transformed cell lines were compared for growth in Eagle medium with 2 or 10% fetal calf serum, colony formation in soft-agar culture, and tumor growth in rats transplanted with the transformed cells. Several transformed cell lines of E1- 4Y and E1-3- 4Y cells showed colony formation in soft-agar culture and abundant expression of the E1B gene. T antigen f was seen by immunofluorescence as flecks in these cells, in which the E4 gene was transcribed, but was not seen in E1Y cells, suggesting that T antigen f was encoded by the E4 gene. The suggestion was confirmed by the observation that T antigen f was detected in COS-1 cells transfected singly with gE4 DNA by immunofluorescence with polyclonal and monoclonal antibodies. Transcription of the E4 gene was confirmed in gE4 -transfected COS-1 cells. T antigen f, one of the E4 gene products, was identified as a polypeptide of molecular weight 11,000 (E4- 11K ) by immunoprecipitation with monoclonal antibodies. The above results also suggest that expression of the E4 gene gives cells the advantage of forming colonies in soft-agar culture. A tendency was noticed for E1B gene expression to be enhanced by E4 gene expression. The relationship between enhancement of colony formation in soft-agar culture and enhancement of E1B gene expression is discussed.

Adenoviruses, Human↗
PubMed1983

mRNA species and proteins of adenovirus type 12 transforming regions: identification of proteins translated from multiple coding stretches in 2.2 kb region 1B mRNA in vitro and in vivo.

Analysis of cytoplasmic RNA in Ad12-infected and -transformed cells showed that more than 20 mRNA species are transcribed from regions 1A and 1B. These mRNA species could be classified into four classes according to the extent to which they were expressed in infected and transformed cells under various conditions. The proteins synthesized from the major species of each of these mRNAs were identified by in vitro translation. In region 1A, the 40K (and 38K) proteins were synthesized from three mRNA species that differed from one another in their 5' ends, and in region 1B three proteins with molecular weights of 19,000 (19K), 50K, and 17K were synthesized from a single species of 2.2 kb mRNA. Only the 19K protein was immunoprecipitated by anti-T serum (G serum) from rats bearing GY1 cell tumors, [GY1 cells are rat cells transformed by the Ad12 HindIII-G fragment (0-6.8 map units).] The 19K protein was also detected by immunoprecipitation in extracts from both transformed and infected cells. The 17K protein was immunoprecipitated by anti-T serum (C serum) derived from rats bearing CY1 cell tumors, but was not by G serum. [CY1 cells are rat cells transformed by the Ad12 EcoRI-C fragment (0-16.5 map units).] The 17K protein was also translated in vitro from the 0.5 kb region 1B mRNA. These results suggest that the 19K and 17K proteins correspond to Ad12 T antigen f and polypeptide IX, respectively, and that these two proteins are translated in vivo from different coding regions in 2.2 kb mRNA in CY1 cells.

Adenoviruses, Human↗
PubMed1983

The 19-kDal protein encoded by early region 1b of adenovirus type 12 is synthesized efficiently in Escherichia coli only as a fused protein.

We have constructed recombinant plasmids that direct the synthesis of the Mr 19 000 protein encoded by the adenovirus type 12 (Ad12) E1b region as either a native protein or a protein fused to the amino-terminal portion of the elongation factor EF-TuB in Escherichia coli cells. Using these recombinants, we could synthesize a large amount of the fused protein, while only a small amount of the native Mr 19 000 protein was produced. The failure to synthesize the native Mr 19 000 protein in E. coli cells was ascribed to inefficient translation.

Adenoviridae↗
PubMed1983

Antigenic and biochemical characterization of poliovirus type 1 isolates of non-vaccine origin.

During the past 15 years, five poliovirus type 1 strains with non-vaccine-like antigenicity have been isolated in Japan. Of these isolates, two were from paralytic poliomyelitis patients not associated with the use of Sabin vaccine, and three were apparently introduced from abroad. All the isolates could be readily distinguished from the corresponding Sabin type 1 vaccine strain by oligonucleotide mapping of the viral RNA and by polyacrylamide gel electrophoresis of the viral proteins. The oligonucleotide map of the virulent Mahoney strain which has non-vaccine-like antigenicity was very similar to the map of Sabin type 1 strain. These data indicate that none of the isolates were derived from Sabin type 1 vaccine or its parental Mahoney strain. In addition, some isolates had close antigenic relationship with one another. It is probable that all these strains were introduced from foreign lands where wild poliovirus strains are prevalent.

Animals↗
PubMed1983

Expression of adenovirus type 12 E1A gene in monkey cells, using a simian virus 40 vector.

Simian virus 40 (SV40) recombinants carrying the adenovirus type 12 E1A gene were constructed. The SV40 expression vector was constructed by removing most of the VP1 gene and an internal part of the intervening sequence for late 16S RNA and by joining the 5' and 3' splice sites into a small segment. The adenovirus type 12 E1A gene with or without its own promoter was inserted downstream from the SV40 late promoter and the splicing junctions. The recombinant DNA was propagated and packaged in monkey cells by cotransfection with an early temperature-sensitive mutant (tsA58) DNA as helper. Immunofluorescent staining of the monkey cells infected with the resulting virus stocks showed that up to 20% of the cells overproduced the E1A gene products in the nuclei. Two-dimensional gel electrophoresis of the products indicated that the products were very similar or identical to the authentic polypeptides synthesized in adenovirus type 12-infected human embryo kidney cells. The E1A mRNA was initiated at the SV40 late promoter irrespective of the presence of the E1A promoter and terminated at either the E1A or the SV40 polyadenylation signal. These hybrid mRNAs were correctly spliced in the E1A coding region.

Adenoviruses, Human↗
PubMed1983

Hyperproduction of adenovirus type 12 E1B gene product in monkey cells, using a simian virus 40 vector.

Simian virus 40 recombinant DNAs carrying the adenovirus type 12 E1B gene were constructed, propagated, and packaged in monkey cells. Monkey cells infected with the resulting virus stocks hyperproduced the E1B gene products in more than 80% of the cells as revealed by immunofluorescence. The products were distributed in both the nuclei and the cytoplasm, and a condensed form of fleck structure was observed in the cytoplasm. Polyacrylamide gel electrophoresis of the cell extracts and their immunoprecipitates detected the E1B-coded 19,000-molecular-weight protein but not the 50,000-molecular-weight protein. The 19,000-molecular-weight protein and the simian virus 40 VP1 protein were synthesized in nearly equal amounts.

Adenoviruses, Human↗
PubMed1983

Expression of adenovirus type 12 early region 1 in KB cells transformed by recombinants containing the gene.

The adenovirus type 12 (Ad12) early region 1 (E1) gene was introduced into KB cells by using a dominant selection vector, pSV2-gpt, and over 80 Gpt+ KB cell clones were established. Three types of recombinant DNAs (gAE1A, gARC, and gABA) were constructed. They contained the AccI-H, EcoRI-C, and BamHI-A fragments, respectively, of Ad12 DNA in pSV2-gpt. Five of 50 (10%) gABA-transformed cell clones, 12 of 18 (67%) gAE1A-transformed cell clones, and 10 of 18 (56%) gARC-transformed cell clones complemented the growth of Ad5 dl312 (deletion in E1A) and were designated as Gpt+ Ad+ cell clones. In these cell clones at their early passages, recombinant genome sequences were detected in cellular DNA and were expressed. T antigen g (the E1A gene product) was detected by immunofluorescence. The Gpt+ Ad+ cell clones supported the growth of Ad5 deletion mutants in parallel with the expression of Ad12 E1A or E1A plus E1B genes. After infection of Gpt+ Ad+ cell clones with Ad5 dl312, the early genes of dl312 were efficiently transcribed, indicating the expression of the pre-early function of the Ad12 E1A gene. Two clones each from gAE1A-,gARC-, and gABA-transformed cells were subcultured for a long period to determine the stability of the transfecting DNAs. Subculture in a nonselective medium resulted in cells which lost the transfecting DNAs. Subculture in a selective medium resulted in the selection of cells which maintained the gpt gene expression but lost the Ad12 gene expression. These results indicate that the transfecting DNA is present in an unstable state in KB cells.

Adenoviruses, Human↗
PubMed1983

Isolation of a nondefective recombinant between adenovirus type 5 and early region 1A of adenovirus type 12.

A nondefective recombinant between adenovirus type 5 (Ad5) and type 12 (Ad12), rc-1 (Ad5 dl312, carrying the Ad12 E1A gene), was isolated from hamster cell foci transformed by a defective recombinant, rcB-1 (dl312, carrying the Ad12 E1 gene). The recombinant rc-1 grew in human embryo kidney and KB cells in the absence of helper and synthesized Ad12 T antigen g, the product of the E1A gene. The genome of rc-1 has a deletion between 79.9 and 82.5 map units of Ad5 dl312 DNA with an insertion of 0.1 to 5.5 map units of Ad12 DNA at the deletion site. The mRNAs of Ad12 E1A were transcribed from the Ad12 E1A promoter, and unusual RNAs were abundantly transcribed from the Ad5 E3 promoter on the opposite strand. The frequency of cell transformation with rc-1 was lower than those with Ad5 and Ad12 wild types.

Adenoviruses, Human↗
PubMed1982

Alteration in oligonucleotide fingerprint patterns of the viral genome in poliovirus type 2 isolated from paralytic patients.

A close relationship was demonstrated by oligonucleotide fingerprinting between genomes of the poliovirus type 2 Sabin vaccine strain and recent isolates from paralytic cases associated with vaccination in Japan. The oligonucleotide maps of isolates from an agammaglobulinemic patient, who continued to excrete poliovirus type 2 for 3.5 years after the administration of oral vaccine, showed that the genomic alteration proceeded gradually, retaining the majority of the oligonucleotides characteristic of the vaccine strain for a long period, indicating vaccine origin for the isolates. The final isolate at month 41, however, lost the majority of these oligonucleotides. The heterologous antigenic relationship between the final isolate and the previous isolates was also observed. The serial alteration in electrophoretic mobility of the major structural proteins (VP1, VP2, and VP3) was observed throughout the excreting period. These results indicate that the population of the virus in this individual changed markedly during the last short period (about 3 months), in which the treatment with secretory immunoglobulin A was carried out. Genome comparisons in oligonucleotide maps show that some oligonucleotides in the genome of the vaccine strain are highly mutable after passage in humans.

Antigens, Viral↗
PubMed1982

Dependence of tumor-forming capacities of cells transformed by recombinants between adenovirus types 5 and 12 on expression of early region 1.

Recombinants between an adenovirus type 5 (Ad5) deletion mutant and the Ad12 DNA fragment containing early region 1 (E1) were isolated from cells cotransfected with the EcoRI-C fragment of Ad12 DNA and Ad5 dl312 (deletion in E1A) DNA (rcA) and from cells cotransfected with the SalI-C fragment of Ad12 DNA and Ad5 dl312 DNA (rcB). No recombinant was isolated from cells cotransfected with Ad5 dl313 (deletion in E1B) DNA and restriction fragments of Ad12 DNA. Both rcA and rcB are defective and able to replicate in human embryo kidney (HEK) and KB cells with complementation by dl312. Both rcA and rcB formed Ad12 T antigen g, but not T antigen f, in infected HEK and KB cells. In rcA- and rcB-infected cells, Ad5 E1B and Ad12 E1A genes are transcribed. Heteroduplex and size analyses of rcA-1 or rcB-1 DNA fragments hybridized with Ad12 DNA revealed that rcA-1 DNA has a deletion between 5 and 15 map units with an insertion of a portion of Ad12 DNA (10%) and that rcB-1 DNA has a deletion between 70 and 80 map units with an insertion of a portion of Ad12 DNA (10%). The transformed cell lines, RCAY and RCBY, were established after infection of rat 3Y1 cells with rcA and rcB, respectively. Both Ad5 and Ad12 DNA sequences are contained in these cells. In RCAY cells, Ad12 T antigen g is detected, but Ad12 T antigen f is not. In RCBY cells, both Ad12 T antigen g and f are detected. Only the Ad12 E1A gene is transcribed in RCAY cells, whereas Ad5 E1B, Ad12 E1A, and Ad12 E1B genes are transcribed in RCBY cells. In soft-agar cultures, RCBY cells form large colonies, whereas RCAY cells form only tiny colonies. RCBY cells form tumors as efficiently as 12WY cells in transplanted rats. RCAY cells formed tumors inefficiently. Ad5-transformed 5WY cells do not form tumors. These observations indicate that the efficient tumor formation by RCBY cells is dependent on the expression of the Ad12 E1A and E1B genes, whereas the inefficient tumor formation by RCAY cells is due to the expression of only the Ad12 E1A gene.

Adenoviruses, Human↗
PubMed1981

Nucleotide sequence of the transforming early region E1b of adenovirus type 12 DNA: structure and gene organization, and comparison with those of adenovirus type 5 DNA.

The nucleotide sequence of the entire transforming early region of E1b of the highly oncogenic adenovirus type 12 (Ad12) DNA has been determined. The total sequence (3860 base pairs) encompasses the entire transforming early region E1 of Ad12 DNA. From the sequence for the E1b region of Ad12, and the transcription map of the E1b region (1, 2, 3, and this paper) the structure and gene organization of the early region E1b of Ad12 DNA were analyzed and compared with those of the E1b region in the non-oncogenic Ad5 DNA (4, 5). Most of the sequences in the E1b region of Ad12 was highly homologous to that of Ad5. It is predicted that the Ad12 region E1b codes for polypeptides of 53.9, 19.1, and 8.9 kd. This situation is identical with that of the Ad5 region E1b which codes for polypeptides of 54.9, 20.6, and 8.3 kd. The function of these predicted polypeptides encoded by the E1b regions in cell transformation is discussed.

Adenoviridae↗
PubMed1981

Incomplete transformation of rat cells by a deletion mutant of adenovirus type 5.

Rat 3Y1 cells were infected with adenovirus type 5 (Ad5) wild type, dl312 (deletion of 902 base pairs between 1.5-4.5 map units), and dl313 (deletion of 2,350 base pairs between 3.5-10.5 map units). After cultivation for 4 weeks, transformed foci appeared in wild type- and dl313-infected cells. No focus was observed in dl312- and mock-infected cells. Foci induced by dl313 were less dense than those induced by wild type. Cell lines (313Y cells) established from dl313-induced foci contained the E1 gene of the dl313 genome (E1a only). Cell lines (5WY cells) established from Ad5 wild type-induced foci contained the E1 gene of wild type (E1a and b). The difference between the transcriptional patterns of the E1 gene in 313Y cells and that in 5WY cells was the same as the difference in dl313- and wild type-infected cells. Colonies were formed in soft agar culture inoculated with 5WY cells, but no colony was formed after inoculation of 313Y cells. The transformed phenotype of 313Y cells was incomplete compared with that in 5WY cells. In nongrowing 3Y1 cells, dl313 and Ad5 wild type induced cellular DNA synthesis but dl312 did not. The above results suggest that the E1a gene is functioning in dl313-infected but not in dl312-infected cells and that such functions as induction of cellular DNA synthesis and transformation of cells are dependent on expression of the Ad5 E1a gene.

Adenoviruses, Human↗
PubMed1980

Chromosomal alterations of rat cell lines transformed by human adenovirus type-12 virion, whole DNA and left-end DNA fragments.

A normal rat cell line, 3Y1-B clone 1-6 (3Y1) and its adenovirus (Ad) type-12-transformed derivatives, W4 (transformed by Ad12 Virion), WY3 (transformed by Ad12 whole DNA), CY1-1 (transformed by the Ad12 EcoRI-C fragment, left 16.5%), GY1-1 (transformed by the Ad12 HindIII-G fragment, left 6.8%) and HY1 (transformed by the Ad12 Acd-H fragment, left 4.7%) were studied cytogenetically. 3Y1 and some of the transformed cell lines (W4, WY3 and GY1-1) were diploid or pseudodiploid, while others (CY1-1 and HY1) were hypotetraploid. A metacentric marker M1 was detected in GY1-1 cells and another marker M2 in W4 and CY1-1 cells at a high frequency. By the Giemsa banding technique, the metacentric markers M1 and M2 from these fully transformed cell lines were identified as isochromosomes derived from 1q (M1) and 3q (M2), respectively. On the other hand, the markers were detected only at a low frequency in incompletely transformed HY1 cells. However, hypersomy in chromosome No. 1 was observed at a high frequency in this cell line. It can be concluded that hypersomy of chromosomes No. 1 or 3 found in transformants and metacentric markers found in complete transformants are characteristic features in rat cells transformed by Ad12.

Adenoviruses, Human↗