Isolation and identification of the Aspergillus nidulans gdhA gene encoding NADP-linked glutamate dehydrogenase

Summary The Neurospora crassa am gene was used as a heterologous probe to identify clones from two independently constructed Aspergillus nidulans gene libraries. These clones have a common HindIII 1.85 kb fragment. This A. nidulans nucleotide stretch hybridises to a N. crassa 2.7 kb BamHI fragment of wild type DNA but not to a co-migrating fragment from the DNA of the N. crassa am132 deletion mutant. One A. nidulans clone was shown to complement the N. crasse am132 deletion strain. The N. crassa transformants show low levels (5%) of heterologous glutamate dehydrogenase activity. The A. nidulans gdhA gene was found to locate in N. crassa at both the homologous (i.e. am) site as well as non-nomologous sites. Partial nucleotide analysis of the fragment has revealed the 5 end of the locus and considerable homology with the N. crassa am gene. We concluded that we have cloned the A. nidulans gdhA gene.Abbreviations bp base pairs - kb 1,000 bp - GDH NADP-Iinked glutamate dehydrogenase - NADP nicotinamide adenine dinuc leotide phosphate - SDS sodium dodecyl sulfate - phage lambda - DTT dithiothreitol - SSC 0.15 M NaCl, 0.015 Na₃ citrate pH 7.0     

Molecular Ecology and Evolution of Streptococcus thermophilus Bacteriophages—a Review

Bacteriophages attacking Streptococcus thermophilus, a lactic acid bacterium used in milk fermentation, are a threat to the dairy industry. These small isometric-headed phages possess double-stranded DNA genomes of 31 to 45 kb. Yoghurt-derived phages exhibit a limited degree of variability, as defined by restriction pattern and host range, while a large diversity of phage types have been isolated from cheese factories. Despite this diversity all S. thermophilus phages, virulent and temperate, belong to a single DNA homology group. Several mechanisms appear to create genetic variability in this phage group. Site-specific deletions, one type possibly mediated by a viral recombinase/integrase, which transformed a temperate into a virulent phage, were observed. Recombination as a result of superinfection of a lysogenic host has been reported. Comparative DNA sequencing identified up to 10% sequence diversity due to point mutations. Genome sequencing of the prototype temperate phage Sfi21 revealed many predicted proteins which showed homology with phages from Lactococcus lactis suggesting horizontal gene transfer. Homology with phages from evolutionary unrelated bacteria like E. coli (e.g. lambdoid phage 434 and P1) and Mycobacterium L5 was also found. Due to their industrial importance, the existence of large phage collections, and the whole phage genome sequencing projects which are currently underway, the S. thermophilus phages may present an interesting experimental system to study bacteriophage evolution.     

Identification of the yeast DNA polymerase I gene with antibody probes

Summary Partially overlapping fragments of the gene encoding yeast DNA polymerase I have been cloned by immunological screening of a yeast genomic library constructed in the phage expression vector gt11. The three gene fragments we analyzed in detail encode part of a yeast protein that has been identified as yeast DNA polymerase I, because it shares with this enzyme a number of antigenic determinants. In fact, the yeast protein fragments expressed by the recombinant phages react with both polyclonal and monoclonal antibodies raised against different, highly purified preparations of DNA polymerase I. Moreover, they can be used to affinity purify antibodies specifically reacting with active DNA polymerase I polypeptides and they compete with the yeast enzyme for binding to antibodies that inhibit catalytic activity. The gene is located on chromosome XIV in the yeast genome, and it is transcribed as a 5.2 kb mRNA.     

Cloning of a functional human adenine phosphoribosyltransferase (APRT) gene: Identification of a restriction fragment length polymorphism and preliminary analysis of DNAs from APRT-deficient families and cell mutants

A complete human APRT gene has been isolated from a lambda phage genomic library using cloned mouse APRT DNA as a probe. The human gene, contained in a recombinant lambda phage designated Huap15, is functional by virtue of its capacity to transfer human APRT activity to Aprt^– mouse recipient cells after phage-mediated transfection. Digestion of Huap15 DNA with BamH1 generated a 2.2-kb fragment that is the only fragment of eight produced to hybridize with the mouse APRT gene. This 2.2-kb BamH1 fragment is a unique, single copy sequence, and has been used to identify a restriction fragment length polymorphism (RFLP) associated with the APRT locus. Taq1 digestion and Southern blot analysis of DNAs from 49 unrelated individuals produced three different patterns. DNAs of 30 individuals produced a restriction pattern of three labeled fragments about 500 bp, 600 bp, and 2.1 kb in size, which is characteristic for individuals homozygous for the more common allele. Two individuals homozygous for the less frequent allele displayed labeled fragments of 500 bp and 2.7 kb. The remaining 17 DNA samples produced all four labeled bands as expected for heterozygous individuals. The frequency of heterozygotes in the population is about 35%, while the frequency of the less common allele is about 0.21. Restriction enzyme analysis of DNAs from two APRT-deficient brothers and from an unrelated heterozygote revealed no gross deletions or rearrangements, nor the Taq1 polymorphism.     

Effect of the combined action of rimantidine and dimethylsulfoxide on phage reproduction

The effect of dimethylsulfoxide (DMSO) and rimantidine on the reproduction of phages T-3, T-4, T-7, and were studied singly and in combination. Maximal activation of reproduction of phages T-3 and T-7 by DMSO was observed during the first 6 min of the latent period. Meanwhile this compound had no action on reproduction of phages T-4 and , a result attributable to the use of intracellular RNA polymerase by these phages. DMSO was shown to stimulate synthesis of the DNA of phage T-3, evidently through its action on phage RNA polymerase. During the combined action of these compounds, partial blocking of their mutually opposite action on the synthesis of phage macromolecules and total abolition of the inhibitory action of remantidine on the yield of infectious phage T-3 were observed.Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 7, pp. 839–840, July, 1976.     

Virostatic action of rimantidin studied on a phage-bacterium model

The action of rimantidin on reproduction of phages T3, T4, and was investigated. The maximal inhibitory action of the compound was observed between the first and sixth minute of infection with phage T3, but no such effect was observed on replication of phages T4 and . It is postulated on the basis of the results of incorporation of labeled precursors into phage DNA, RNA, and protein and the character of manifestation of the action of the compound on the phage yield that the inhibitory effect of rimantidin is evidently determined by its effect on bacterial and phage RNA polymerases, mainly on the latter.Research Laboratory of Experimental Immunobiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 5, pp. 564–566, May, 1970.     

Transformation of Saccharomyces cerevisiae with plasmids containing fragments of yeast 2 μ DNA and a suppressor tRNA gene

Hybrid plasmids have been constructed containing segments of the yeast plasmid 2 DNA, the yeast ochre-suppressing SUP4.0 gene and the bacterial plasmid pBR322. Yeast transformation is detected with a host containing multiple ochre auxotrophic mutations. The transformed SUP4.0 gene is active and can promote growth in the absence of all the requirements. Plasmids containing different fragments of 2 DNA all appear to be active in high frequency transformation of yeast containing 2 DNA, except those containing the HindlII-D fragment. The transforming plasmids undergo recombination with the indigenous 2 DNA. Integration of the transforming plasmid into the host chromosome has been detected by hybridization of restriction enzyme cleaved DNA with labelled pBR322. The plasmids contain restriction enzyme sites which can be used for cloning other genes into yeast.Abbreviations kb kilobase pair - 2 the yeast plasmid of 6.2 kb size     

Monoclonal antibodies reactive with specific amino acid sequences of the 126K protein of tobacco mosaic virus

Summary A structure/function study has been initiated for the ³⁴ bacteriophage proteins involved in lysogeny inSalmonella newington. Hydroxylamine and nitrosoguanidine mutagenesis of a wild type ³⁴ phage was used to generate clear plaque variants. Complementation analysis was used to define four genes involved in the phage lysogenic pathway. A relative mapping order has been established. In addition, a virulent mutant, ³⁴vir82, which defines a repressor binding site has been isolated.Dedicated to the memory of Professor Harrison Hatch Echols of the Department of Molecular and Cell Biology of the University of California at Berkeley.     

Sequence Analysis of the 5′-Flanking Region of the Gene Encoding Human HOXA-7

We have isolated and characterized the immediate 5-flanking region (886 bp) of the gene encoding human HOXA-7. When the total sequence was compared with those of mice, 93% of the 3 518 bp (nt 370–886) sequences were identical, in which the 245 bases just preceding theAUG initiator codon (nt 614) was as highly conserved as in the coding region (nt 614–886). Sequences further upstream (nt 1–370) by comparison were highly diverged. In the 245 bp region, 8 stop and 3 initiation (including the initiator) codons were located, and a 50-aa long presumptive polypeptide was encoded. Nucleotide sequence analysis revealed three Spl and oneAP2 binding sites, as well as one CAATbox. However, there was no consensus sequence for a TATA box in the 5 flanking region. One RARE repeat, one krox20 and three Hox-PBC binding sites were detected. Since many of the factor recognition sites were located in the immediate 5 flanking sequences of a highly-conserved region, it might be speculated that a regulatory mechanism for Hox gene expression is conserved throughout the evolution and one possible mechanism could be at the post-translational level.     

Distinct mechanism of human neuroblastoma cell adhesion to fibronectin

We investigated the adhesion of three morphologically distinct human neuroblastoma cell lines (NCG, GOTO and SK-N-DZ) to intact fibronectin, central cell binding domain fragment (CBF) and CS peptide-IgG conjugates in the fibronectin molecule. Each cell line was found to express different integrin fibronectin receptors ( _{3 1}, _{4 1} and ₅₁), although similarly attached on intact fibronectin. To CBF, NCG attached well, while GOTO moderately and SK-N-DZ poorly attached. Only GOTO adhered to CS1-IgG. RGDS inhibited the spreading of NCG and SK-N-DZ on intact fibronectin, but it barely inhibited that of GOTO. The analysis by fluorescence-activated cell sorting (FACS) revealed that NCG expressed abundant ₃₁ and ₅₁, but little ₄₁, while GOTO expressed a large amount of ₄₁ as well as ₅₁. SK-N-DZ was undetectable in any of these molecules, but expressed _v1, which was identified by immunoprecipitation and immunoblotting. Polyclonal antibody to _v3 inhibited the adhesion of SK-N-DZ but not that of NCG or GOTO on intact fibronectin. These results suggest the existence of a distinct mechanism of cell adhesion to fibronectin among human neuroblastoma cell lines. It remains to be determined if such heterogeneous adhesion properties are related to the unique metastatic character of human neuroblastoma.     

Expression of the cloned endo-1,3-1,4-β-glucanase gene of Bacillus subtilis in Saccharomyces cerevisiae

Summary A cloned endo-1,3-1,4--glucanase gene from the Gram-positive bacterium B. subtilis has been located by deletion analysis on a 1.4 kb PvuI-ClaI DNA fragment. This gene has been sub-cloned in the yeast LEU2 vector pJDB207 to produce a hybrid plasmid designated pEHB9. pEHB9 has been transformed to S. cerevisiae and shown to direct the synthesis of an endo-1,3-1,4--glucanase in yeast. The -glucanase activity was low and could only be detected in crude cell extracts of yeast harbouring pEHB9.     

Cloning,Sequencing, Expression and Promoter Analysis of a Structural Protein of Bacteriophage MB78

Bacteriophage MB78, a virulent phage of Salmonella typhimurium isolated in our laboratory. It is different from the well-known temperate phage P22 and 9NA. A detailed physical map has been constructed. To understand more about the physiology and genetics of this interesting phage it has become necessary to fragment the phage genome, clone the fragments and analyze in depth. A number of promoters of bacteriophage MB78 have been cloned and characterized recently. As a part of this program, in this investigation, we report cloning, sequencing and expression and promoter analysis of the ClaI G fragment. We identified the expressed protein as phage structural. Phage structural proteins play a vital role in forming the core head of the phage particle.     

Identification of an autonomously replicating sequence near a histone gene of Physarum polycephalum

Summary Fragments of DNA with function as autonomous replication sequences in yeast were cloned from Physarum polycephalum. The ars activity is located in a 1.2 kbp fragment extanding 1.5 kbp to 2.7 kbp upstream of the 5 end of a histone H4 gene. Our recent finding that a replication origin is located at a distance less than 3 kbp of this histone gene suggests that the ars element identified coincides with a specialized replication origin and can be used to direct chromosome replication in Physarum polycephalum.     

Structural analysis of proviral DNA in simian foamy virus (LK-3)-infected cells

Summary Proviral DNA of the T-cell lymphotropic simian foamy virus strain LK-3 was characterized. In infected cells, multiple copies of unintegrated linear duplex viral DNA of about 13 kbp length are present. Nuclease S1 treatment of the DNA generated two fragments of 6.5 and 6.0 kbp length that were cloned in phage and plasmid vectors. The proviral DNA contains a single-stranded gap of 109 nucleotides. A DNA fragment spanning the gap was cloned after completing the double strand by DNA synthesis in vitro. At the 3 end, the gap contains a polypurine tract (PPT) similar to the putative initiation site of retroviral plus strand DNA synthesis, suggesting discontinuous DNA synthesis. Further analysis of the genome architecture revealed LTRs of 1.7 kbp length. An additional 1.7 kbp DNA fragment was detected after nuclease S1 digestion of proviral DNA and probably represents trimmed intermediates of strong-stop DNA.     

Molecular cloning and characterization of a Candida tsukubaensis α-glucosidase gene in the yeast Saccharomyces cerevisiae

Summary The molecular cloning of an -glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cerevisiae is reported. The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S. cerevisiae. The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing -1,2, -1,3, -1,4 and 1,6 linked, as well as aryl and alkyl, d-glucosides. On the basis of its substrate specificity profile, the cloned enzyme was classified as an -glucosidase (E.C. 3.2.1.20). It has a pH optimum in the range 4.2–4.6, a temperature optimum of 58°C and is readily inactivated at pasteurization temperature (60°C). Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts. A rapid plate-assay, based on the utilization of a chromogenic substrate X--d-glucoside to detect the expression of the cloned -glucosidase in S. cerevisiae transformants, was developed.     

Detection of deletion mutations extending beyond the HPRT gene by multiplex PCR analysis

A multiplex PCR assay was developed for the rapid analysis of deletion size at the hypoxanthine phosphoribosyltransferase (hprt) locus. The DNA sequence of mapped DNA segments flanking thehprt gene was determined. These cloned DNAs were derived from the ends of a set of overlapping yeast artificial chromosomes (YAC) defining a contig of 8 Mb at Xq26 and includinghprt. We used bubble PCR to isolate an additional YAC end-clone. Seven primer pairs were derived from DNA sequence analysis of the clones and incorporated into a multiplex PCR assay. These primer pairs define loci located approximately 750 kb and 350 kb upstream ofhprt and 300 kb, 540 kb, 900 kb, 1260 kb, and 1400 kb downstream ofhprt. A primer pair for an unlinked and unselected gene sequence (K-ras) was also included in the multiplex reaction to serve as an internal positive control. Using this new assay,hprt mutant DNAs can be screened to determine the extent of deletion. Deletions larger than 2 Mb have been identified and show that large deletions can be tolerated at this hemizygous locus.     

Polymorphism in the 3′-untranslated region of the thymidylate synthase gene and sensitivity of stomach cancer to fluoropyrimidine-based chemotherapy

To investigate the relationship between polymorphism in the 3-untranslated region (3-UTR) of the thymidylate synthase (TS) gene and sensitivity of gastric cancer to 5-fluorouracil (5-FU)-based chemotherapy, 106 cases of advanced gastric cancer were analyzed. All patients were treated with 5-FU-based chemotherapy; DNA from peripheral blood leukocytes was obtained before therapy. TS 3-UTR genotypes were detected by PCR-RFLP. Polymorphism in the TS 3-UTR can be classified into three groups according to the presence or absence of a 6 bp nucleotide fragment: the –6/–6 bp, –6/+6 bp and +6/+6 bp groups. The response rate of the –6/–6 bp and –6/+6 bp groups was found to be significantly higher than the +6/+6 bp group. These results show that the presence of the TS 3-UTR 6 bp nucleotide fragment can be correlated with the sensitivity of gastric cancer to 5-FU-based chemotherapy, and that the TS 3-UTR polymorphism profile can be used to guide the choice of 5-FU-based chemotherapy in advanced gastric cancer.     

Trehalose and maltose metabolism in yeast transformed by a MAL4 regulatory gene cloned from a constitutive donor strain

Summary A 6.8 kb fragment of DNA containing the regulatory sequence MAL4p has been cloned from a genomic library prepared from Saccharomyces cerevisiae strain 1403-7A which ferments maltose constitutively. The library was prepared by ligation of 5–20 kb Sau3AI restriction fragments of total yeast DNA into the BamH1 restriction site of shuttle vector YEp13. A restriction map of the cloned fragment indicates that it encompasses a 2.6 kb segment which closely resembles the regulatory MAL6 gene previously identified (Needleman et al. 1984). The hybrid plasmid, p(MAL4p)4, could transform maltose-nonfermenting strains which contain cryptic -glucosidase and maltose permease genes (malp MALg), but could not transform strains containing a functional regulatory sequence and a defective maltase-permease region (MAlp malg). A correlated absence of maltase and permease DNA from the cloned fragment was indicated by the restriction map. Although the cloned DNA fragment was derived from a constitutive strain, maltose fermentation and -glucosidase formation by yeast transformed with p(MAL4p)4 was largely inducible by maltose and sensitive to catabolite repression. Moreover, the active trehalose accumulation pattern (TAC(+) phenotype) linked to the complete MAL4 locus in strain 1403-7A and other constitutive MAL strains (Oliveira et al. 1981b) was not found in p(MAL4p)4 transformants. It may be concluded that constitutivity of maltose fermentation and the associated active trehalose accumulation are not merely consequences of a cis-dominant mutation causing constitutive formation of the MALp regulatory product. Moreover, constitutivity may not be caused solely by a mutation within the structural region of the MALp gene.     

Cloning of the thymidylate synthetase gene (thyPIG 3) from theBacillus subtilis temperate phage IG 3

Summary ThethyPIG 3 gene fromBacillus subtilis bacteriophage IG 3 was cloned in the plasmid pHV 33. Two recombinant plasmids, pISL 61 and pISL 62 carrying that gene are effective in transforming to thymine prototrophy bothEscherichia coli (by complementation) andB. subtilis (by complementation and recombination). The comparison of cloned fragment containing thethyPIG 3 gene and thethyP 3 gene from phage 3 T, by restriction analysis and DNA hybridization, suggests a strong homology between the two. ThethyPIG 3 gene was mapped in this study in the central region of the IG 3 genome.     

Effects of interleukin-1β and tumor necrosis factor-α on osteoblastic expression of osteocalcin and mineralized extracellular matrix in vitro

Osteoblasts play a pivotal role during the bioresponse of bone to agents that stimulate bone resorption and/or inhibit bone formation including hormones, polypeptide growth factors, and cytokines. We examined the cytokines interleukin-1-beta (IL-1) and tumor necrosis factor-alpha (TNF-) for their effects on osteoblastic proliferation and development and expression of alkaline phosphatase and the osteoblast-specific protein osteocalcin in a mineralizing environment. Primary rat osteoblast-like cells (ROB) and osteoblastic cell lines derived from rat (ROS 17/2.8) and human (MG-63) osteosarcomas were studied. IL-1 and TNF- were chosen because of their critical importance during the host response to local inflammatory stimuli. Qualitatively similar two- to threefold inhibition of osteocalcin synthesis by IL-1 and TNF- were observed in all three postconfluent bone-forming model systems. Because of the readily measurable concentrations of osteocalcin produced in our culture protocol, it was not necessary to enhance osteoblastic synthesis of osteocalcin by supplementation with 1,25(OH)₂-vitamin D₃, a treatment which exerts pleiotropic effects on osteoblasts. Under the constraints of our protocol, where alkaline phosphatase and mineralization were already elevated at the 14-day onset of treatment, neither of these phenotypic properties was sensitive to a three-day cytokine exposure. Differences were noted in proliferation, where only TNF- stimulated DNA synthesis in ROB cells, while both cytokines stimulated MG-63 cells. IL-1 and TNF- failed to alter ROS 17/2.8 DNA synthesis except at the highest doses (25 pM IL-1 and l nM TNF-) where inhibition was observed. These results further support the view that cytokine-mediated osteoblastic regulation can be relatively selective.