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We report the complete 5025-base sequence of the human 28S rRNA gene. Variability within the species has been demonstrated by sequencing a variable region from six separately cloned genes. This region is one of three large subunit rRNA regions that show extreme sequence and size variation among species. The interspecies differences suggest species-specific functions for these sections, while the intraspecies heterogeneity indicates differences among ribosomes. Comparison of the human gene with a partial sequence from the chimpanzee 28S gene yields divergence rates for the two species: 0.8% for conserved regions of the gene and 3.7% for a variable region. The rapid divergence rates of variable regions in the ribosomal gene may permit answers to the question of time of separation of closely related species.

The ability of DNA excision-repair processes in diploid human fibroblasts to eliminate potentially cytotoxic and mutagenic lesions induced by UV radiation (254 nm) was demonstrated in two ways: (1) Cells with normal rates of excision were compared with cells with an intermediate rate of excision (XP2BE) and cells with an excision rate less than or equal to 1% that of normal (XP12BE) for sensitivity to the killing and mutagenic action of UV radiation. The normal cells proved resistant to doses of UV which reduced the survival of the XP cells to 14% and 0.7%, respectively, and increased the frequency of mutations to 8-azaguanine resistance in the XP cells 5- to 10-fold over background. (2) Cells in confluence were irradiated with cytotoxic and mutagenic doses of UV and allowed to carry out excision repair. After various lengths of time they were replated at lower densities to allow for expression of mutations to 6-thioguanine resistance and/or at cloning densities to assay survival. Normal cells and XP cells with reduced rates of excision repair (from complementation groups C and D) exhibited a gradual increase in survival from an initial level of 15--20% to 100% if held approximately 20 h in confluence. In contrast, XP12BE cells showed no increase from an initial survival of 20% even when held for 7 days. Normal cells irradiated in confluence but prevented from replicating for 7 days exhibited background mutation frequencies, whereas the mutation frequency in XP12BE cells did not change with the time in confluence.

A recombinant library of double minute chromosomal DNA, enriched in specific sequences that are amplified in Y1 mouse adrenal tumor cells, was used as a source of material to explore the structure and expression of amplified cKi-ras genes in these cells. From DNA sequence analysis of these cloned fragments, we found no evidence for the presence of point mutations previously demonstrated to be associated with activation of the transforming potential of ras genes. A comparison of the mouse gene with that of the homologous human cKi-ras2 gene reveals 94% nucleotide sequence homology within the coding regions and 97% homology for the predicted amino acid composition. Like the human gene, the mouse cKi-ras gene contains alternative 3' coding exons. Blot hybridization analyses of RNA revealed a preferential utilization of the more 3' of the two fourth coding exons in the generation of Y1 cKi-ras transcripts.

Eco-RI-A fragments of the human ribosomal RNA gene family from two types of tissue and three individuals were cloned in lambda vectors and compared by restriction enzyme digestion and electron microscopy. The EcoRI fragment A contains (i) 0.2 kb of the 3' end of the 18S rDNA, (ii) 2.5 kb of internal transcribed spacer and the 5.8S rDNA, and (iii) 4.6 kb of the 28S rDNA gene. All of the six cloned rDNA fragments isolated are identical by these analyses. Moreover, all contain a HincII site that is absent in about 50% of the rDNA identified by genomic blotting. Polymorphism in the nontranscribed spacer rDNA was studied in genomic blots of BamHI-digested DNA, using the 3' end of the 28S rDNA as a probe. The boundaries between the 18S rDNA, internal transcribed spacer, 28s rDNA, and external nontranscribed spacer were determined by R-loop analysis, further defining the organization of the ribosomal RNA precursor.

We have looked for conserved DNA sequences between four herpes simplex virus type 1 (HSV-1) glycoprotein genes encoding gB, gC, gD, and gE and pseudorabies virus (PRV) DNA, HSV-1 DNA fragments representing these four glycoprotein-coding sequences were hybridized to restriction enzyme fragments of PRV DNA by the Southern blot procedure. Specific hybridization was observed only when HSV-1 gB DNA was used as probe. This region of hybridization was localized to a 5.2-kilobase (kb) region mapping at approximately 0.15 map units on the PRV genome. Northern blot (RNA blot) analysis, with a 1.2-kb probe derived from this segment, revealed a predominant hybridizing RNA species of approximately 3 kb in PRV-infected PK15 cells. DNA sequence analysis of the region corresponding to this RNA revealed a single large open reading frame with significant nucleotide homology with the gB gene of HSV-1 KOS 321. In addition, the beginning of the sequenced PRV region also contained the end of an open reading frame with amino acid homology to HSV-1 ICP 18.5, a protein that may be involved in viral glycoprotein transport. This sequence partially overlaps the PRV gB homolog coding sequence. We have shown that the PRV gene with homology to HSV-1 gB encoded the gII glycoprotein gene by expressing a 765-base-pair segment of the PRV open reading frame in Escherichia coli as a protein fused to beta-galactosidase. Antiserum, raised in rabbits, against this fusion protein immunoprecipitated a specific family of PRV glycoproteins of apparent molecular mass 110, 68, and 55 kilodaltons that have been identified as the gII family of glycoproteins. Analysis of the predicted amino acid sequence indicated that the PRV gII protein shares 50% amino acid homology with the aligned HSV-1 gB protein. All 10 cysteine residues located outside of the signal sequence, as well as 4 of 6 potential N-linked glycosylation sites, were conserved between the two proteins. The primary protein sequence for HSV-1 gB regions known to be involved in the rate of virus entry into the cells and cell-cell fusion, as well as regions known to be associated with monoclonal antibody resistance, were highly homologous with the PRV protein sequence. Furthermore, monospecific antibody made against PRV gII immunoprecipitated HSV-1 gB from infected cells. Taken together, these findings suggest significant conservation of structure and function between the two proteins and may indicate a common evolutionary history.

Analysis of six monoclonal antibody-resistant (mar) mutants in herpes simplex virus type 1 glycoprotein B identified two type-common (II and III) and two type-specific (I and IV) antigenic sites on this molecule. To derive additional information on the location of these sites, mar mutations were mapped and nucleotide alterations were identified by DNA sequencing. Each mutant carried a single amino acid substitution resulting from a G-to-A base transition. Alterations affecting antibody neutralization were identified at residues 473, 594, 305, and 85 for mutants in sites I through IV, respectively. Two clonally distinct site II antibodies each selected mar mutants (Gly to Arg at residue 594) that exhibited a reduction in the rate of entry (roe) into host cells. A site II mar revertant that regained sensitivity to neutralization by site II antibodies also showed normal entry kinetics. DNA sequencing of this virus identified a single base reversion of the site II mar mutation, resulting in restoration of the wild-type sequence (Arg to Gly). This finding demonstrated that the mar and roe phenotypes were the result of a single mutation. To further define structures that contributed to antibody recognition, monoclonal antibodies specific for all four sites were tested for their ability to immune precipitate a panel of linker-insertion mutant glycoprotein B molecules. Individual polypeptides that contained single insertions of 2 to 28 amino acids throughout the external domain were not recognized or were recognized poorly by antibodies specific for sites II and III, whereas no insertion affected antibody recognition of sites I and IV. mar mutations affecting either site II or III were previously shown to cause temperature-sensitive defects in glycoprotein B glycosylation, and variants altered in both these sites were temperature sensitive for virus production. Taken together, the data indicate that antigenic sites II and III are composed of higher-order structures whose integrity is linked with the ability of glycoprotein B to function in virus infectivity.

A human cDNA library was screened for sequences homologous to the erbA gene of avian erythroblastosis virus (AEV). One such clone, cHerbA-1, was used to map the chromosomal location of highly homologous human sequences that were found to be present on chromosome 17 as judged by Southern blot screening of a panel of mouse-human hybrid cell lines segregating human chromosomes. cHerbA-1 was hybridized in situ to metaphase chromosomes from a normal male subject and from a female patient with an acute promyelocytic leukemia (APL) having the typical t(15;17) translocation. The results localized the cellular c-erbA sequences on chromosome 17 to the q21-q24 region of normal chromosomes and indicated that the c-erbA sequences remained on the 17q- chromosome in the APL cells, suggesting that they could be assigned to the 17(q21-q22) region. For additional data, we hybridized human neoplastic cells derived from a poorly differentiated acute leukemia carrying a t(17;21) translocation with thymidine kinase (TK)-deficient LMTK- mouse cells. A resulting hybrid, containing only the 21q+ chromosome, did not have human c-erbA sequences. Since the breakpoint on 17q in this translocation was similar to that in the APL t(15;17) translocation, this supported the assignment of c-erbA to the q21-q22 region of chromosome 17. The apparent close proximity of the c-erbA sequences to the chromosomal breakpoints in these two leukemias suggests a possible role for this oncogene homologue in the development of these neoplasms.