The GCR1 requirement for yeast glycolytic gene expression is suppressed by dominant mutations in the SGC1 gene, which encodes a novel basic-helix-loop-helix protein.

The GCR1 gene product is required for maximal transcription of yeast glycolytic genes and for growth of yeast strains in media containing glucose as a carbon source. Dominant mutations in two genes, SGC1 and SGC2, as well as recessive mutations in the SGC5 gene were identified as suppressors of the growth and transcriptional defects caused by a gcr1 null mutation. The wild-type and mutant alleles of SGC1 were cloned and sequenced. The predicted amino acid sequence of the SGC1 gene product includes a region with substantial similarity to the basic-helix-loop-helix domain of the Myc family of DNA-binding proteins. The SGC1-1 dominant mutant allele contained a substitution of glutamine for a highly conserved glutamic acid residue within the putative basic DNA binding domain. A second dominant mutant, SGC1-2, contained a valine-for-isoleucine substitution within the putative loop region. The SGC1-1 dominant mutant suppressed the GCR1 requirement for enolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, and pyruvate kinase gene expression. Expression of the yeast enolase genes was reduced three- to fivefold in strains carrying an sgc1 null mutation, demonstrating that SGC1 is required for maximal enolase gene expression. Expression of the enolase genes in strains carrying gcr1 and sgc1 double null mutations was substantially less than observed for strains carrying either null mutation alone, suggesting that GCR1 and SGC1 function on parallel pathways to activate yeast glycolytic gene expression.

Epidemiology and partial nucleotide sequence of four novel genital human papillomaviruses.

Polymerase chain reaction (PCR)-based genital human papillomavirus (HPV) detection methods that use consensus primers have enabled the broad-spectrum detection of most characterized HPV types. In addition, these techniques have allowed the identification of potentially novel viral sequences in clinical specimens. These methods were used to determine the partial L1 nucleotide sequence (the region generated by L1 consensus primers MY09 and MY11) of four novel viruses. The prevalence of these viruses in cytologically normal and dysplastic cervical specimens and in invasive cervical cancer was also determined. The partial DNA sequences of W13B (MM4), PAP291 (MM7), PAP155 (MM8), and PAP238a (MM9) are most similar to HPV-51, -61, -61, and -34, respectively. Prevalence studies suggest that W13B and PAP238a are cancer-associated, while PAP155 and PAP291 appear to be lower-risk viruses.

Design and analysis of PCR primers for the amplification and cloning of human immunoglobulin Fab fragments.

We have developed PCR primers for the amplification and cloning of the genes encoding human antibody fragments. Two sets of primers were designed to amplify the coding sequence of the complete variable region and the first constant domain of immunoglobulin heavy chains. One set of primers was designed to amplify the coding sequence of complete kappa light chains. These three sets of primers were used in PCR amplifications with cDNA prepared from EBV transformed B cells as the template. The amplified fragments were cloned and their nucleotide sequences were determined. Analysis of the DNA sequences of such PCR generated antibody fragments suggested that our primer sets were able to amplify the major subsets of the heavy and light chain variable region genes. Comparison between the estimated frequency of the different subsets of heavy chain variable region genes and the distribution of our subclones further indicated that one set of our primers was able to proportionally amplify the subsets of the heavy chain family.

Genetic organization of the cellulose synthase operon in Acetobacter xylinum.

An operon encoding four proteins required for bacterial cellulose biosynthesis (bcs) in Acetobacter xylinum was isolated via genetic complementation with strains lacking cellulose synthase activity. Nucleotide sequence analysis indicated that the cellulose synthase operon is 9217 base pairs long and consists of four genes. The four genes--bcsA, bcsB, bcsC, and bcsD--appear to be translationally coupled and transcribed as a polycistronic mRNA with an initiation site 97 bases upstream of the coding region of the first gene (bcsA) in the operon. Results from genetic complementation tests and gene disruption analyses demonstrate that all four genes in the operon are required for maximal bacterial cellulose synthesis in A. xylinum. The calculated molecular masses of the proteins encoded by bcsA, bcsB, bcsC, and bcsD are 84.4, 85.3, 141.0, and 17.3 kDa, respectively. The second gene in the operon (bcsB) encodes the catalytic subunit of cellulose synthase. The functions of the bcsA, bcsC, and bcsD gene products are unknown. Bacterial strains mutated in the bcsA locus were found to be deficient in cellulose synthesis due to the lack of cellulose synthase and diguanylate cyclase activities. Mutants in the bcsC and bcsD genes were impaired in cellulose production in vivo, even though they had the capacity to make all the necessary metabolic precursors and cyclic diguanylic acid, the activator of cellulose synthase, and exhibit cellulose synthase activity in vitro. When the entire operon was present on a multicopy plasmid in the bacterial cell, both cellulose synthase activity and cellulose biosynthesis increased. When the promoter of the cellulose synthase operon was replaced on the chromosome by E. coli tac or lac promoters, cellulose production was reduced in parallel with decreased cellulose synthase activity. These observations suggest that the expression of the bcs operon is rate-limiting for cellulose synthesis in A. xylinum.

Molecular analysis of the yeast VPS3 gene and the role of its product in vacuolar protein sorting and vacuolar segregation during the cell cycle.

vps3 mutants of the yeast Saccharomyces cerevisiae are impaired in the sorting of newly synthesized soluble vacuolar proteins and in the acidification of the vacuole (Rothman, J. H., and T. H. Stevens. Cell. 47:1041-1051; Rothman, J. H., C. T. Yamashiro, C. K. Raymond, P. M. Kane, and T. H. Stevens. 1989. J. Cell Biol. 109:93-100). The VPS3 gene, which was cloned using a novel selection procedure, encodes a low abundance, hydrophilic protein of 117 kD that most likely resides in the cytoplasm. Yeast strains bearing a deletion of the VPS3 gene (vps3-delta 1) are viable, yet their growth rate is significantly reduced relative to wild-type cells. Temperature shift experiments with strains carrying a temperature conditional vps3 allele demonstrate that cells rapidly lose the capacity to sort the vacuolar protein carboxypeptidase Y upon loss of VPS3 function. Vacuolar morphology was examined in wild-type and vps3-delta 1 yeast strains by fluorescence microscopy. The vacuoles in wild-type yeast cells are morphologically complex, and they appear to be actively partitioned between mother cells and buds during an early phase of bud growth. Vacuolar morphology in vps3-delta 1 mutants is significantly altered from the wild-type pattern, and the vacuolar segregation process seen in wild-type strains is defective in these mutants. With the exception of a vacuolar acidification defect, the phenotypes of vps3-delta 1 strains are significantly different from those of mutants lacking the vacuolar proton-translocating ATPase. These data demonstrate that the acidification defect in vps3-delta 1 cells is not the primary cause of the pleiotropic defects in vacuolar function observed in these mutants.

Detection and typing of human papillomavirus in archival cervical cancer specimens by DNA amplification with consensus primers.

We developed a polymerase chain reaction DNA amplification system using two distinct consensus oligonucleotide primer sets for the improved detection and typing of a broad spectrum of human genital papillomavirus (HPV) sequences, including those of novel viruses. The system incorporates one primer set designed to amplify a highly conserved L1 domain and a second primer set designed to amplify a domain within the E6 gene. We used this system to analyze 48 fixed, paraffin-embedded tissue sections (41 specimens from 33 cervical carcinomas, four normal cervical tissues, and several control tissues) for the presence of HPV DNA. HPV sequences were detected in all carcinoma samples and none of the control samples. Hybridization analyses showed that the results obtained with the two amplification schemes concurred completely. This approach allowed rapid confirmation of typing results and may improve the likelihood of detecting a wide variety of HPV sequences, including those of novel HPVs.

The yeast ADR6 gene encodes homopolymeric amino acid sequences and a potential metal-binding domain.

The ADR6 gene of Saccharomyces cerevisiae has an open reading frame which could encode a polypeptide of 1314 amino acids. The predicted mRNA encodes a protein with homopolymeric stretches of asparagine and threonine, particularly near its amino terminus and contains additional sequences consisting of polyglutamine repeats. The predicted protein also contains a potential metal binding (Cys)4-type finger near its carboxy-terminus. An ADR6/beta-galactosidase fusion protein was predominantly nuclear in location, consistent with its role as an activator of ADH2 transcription.