Large non-homology in heteroduplex DNA is processed differently than single base pair mismatches

Summary Unmethylated DNA heteroduplexes with a large single stranded loop in one strand have been prepared from separated strands of DNA from two different strains of bacteriophage , one of which has a 800 base pair IS1 insertion in the cI gene. The results of transfections with these heteroduplexes into wild-type and mismatch repair deficient bacteria indicate that such large non-homologies are not repaired by the Escherichia coli mismatch repair system. However, the results do suggest that some process can act to repair such large non-homologies in heteroduplex DNA. Transfections of a series of recombination and excision repair deficient mutants suggest that known excision or recombination repair systems of E. coli are not responsible for the repair. Repair of large non-homologies may play a role in gene conversion involving large insertion or deletion mutations.     

Molecular cloning and nucleotide sequence of the mutT mutator of Escherichia coli that causes A:T to C:G transversion

Summary The Escherichia coli mutator gene mutT, which causes A:TC:G transversion, was cloned in pBR 322. mutT ⁺ plasmids carry a 0.9 kb PvuII DNA fragment derived from the E. coli chromosome. Specific labelling of plasmid-encoded proteins by the maxicell method revealed that mutT codes for a polypeptide of about 15,000 daltons. The protein was overproduced when the mutT gene was placed under the control of the lac regulatory region on a multicopy runaway plasmid. The nucleotide sequence of the mutT gene was determined by the dideoxy method.Abbreviations Ap ampicillin - IPTG isopropyl--d-thiogalactopyranoside - kb kilobase pair(s) - kDa kilodalton(s) - SDS sodium dodecyl sulphate - Tc tetracycline     

Chimeric evolution of the 2-μm genome in Saccharomyces cerevisiae

We compared the nucleotide substitution pattern over the entire genome of two unique variants of the 6,300-bp selfish DNA (2 m) plasmid in Saccharomyces cerevisiae. The DNA sequence of the left-unique region is identical among 2-m variants, while the right-unique region shows substantial divergence. This chimeric pattern cannot be explained by neutral or Darwinian selection models. We propose that horizontal transmission of the 2-m plasmid coupled with a directed, polarized gene conversion maintains the DNA sequence of the left-unique region, whereas the right-unique region is subject to random drift and Darwinian selection. Correspondence to: G.H. Rank     

The glucose kinase gene of Streptomyces coelicolor and its use in selecting spontaneous deletions for desired regions of the genome

Summary A number of deletions in the glucose kinase (glk) region of the Streptomyces coelicolor chromosome were found among spontaneous glk mutants. The deletions were identified by probing Southern blots of chromosomal DNA from glk mutants with cloned glk DNA. The deletions ranged in size from 0.3 kb to greater than 2.9 kb. When cloned glk DNA was introduced on a C31 phage vector into a glk mutant that contained a deletion of the entire homolgous chromosomal glk region, glucose kinase activity was detected in extracts of these cells. The entire coding information for at least a subunit of glucose kinase is there-fore present on the cloned glk DNA. The 0.3 kb glk chromosomal deletion was used to demonstrate that transfer of chromosomal glk mutations on the the C31::glk phage could occur by recombination in vivo. Since glk mutations frequently arise from deletion events, a method was devised for inserting the cloned glk DNA at sites in the chromosome for which cloned DNA is available, and thus facilitating the isolation of deletions in those DNA regions. C31::glk vectors containing a deletion of the phage att site cannot lysogenize S. coelicolor recipients containing a deletion of the glk chromosomal gene unless these phages contain S. coelicolor chromosomal DNA. In such lysogens, the glk gene becomes integrated into the chromosome by homologous recombination directed by the chromosomal insert on the phage DNA. In appropriate selective conditions, mutants which contain deletions of the glk gene that extend into the adjacent host DNA can be easily isolated. This method was used to insert glk into the methylenomycin biosynthetic genes, and isolate derivatives with deletions of host DNA from within the prophage into the adjacent host DNA. Phenotypic and Southern blot analysis of the deletions showed that there are no genes essential for methylenomycin biosynthesis for at least 13 kb to the left of a region concerned with negative regulation of methylenomycin biosynthesis. Many of the deletions also removed part of the C31 prophage.     

Effects of steroid hormones on age-related expression and modulation of the lysozyme gene of the oviduct of Japanese quail

The expression of lysozyme gene in the oviduct of Japanese quail is age-dependent. Here we show that the expression of the gene is altered by three steroid hormones: 17-estradiol (E), progesterone (P) and glucocorticoid (dexamethasone, G), and their combinations E+P, E+G and P+G. We also show that the levels/affinities of trans-acting factors that bind to specific cis-acting elements in the promoter region of the gene change with age and after steroid administration. These factors are sequence-specific, age and steroid-dependent. It is proposed that administration of appropriate doses of steroid hormones after adulthood may extend the reproductive function and egg laying period in birds.     

The J gene of ?X174: Isolation and characterization of a J gene mutant

Summary The J gene protein of bacteriophage X174 is a component of the mature phage. The previous lack of J gene mutants has prevented an in vivo analysis of J protein functions. A X174 mutant was constructed by inserting an 11 nucleotide sequence into the J gene. This mutant, designated insJ, was viable only in the presence of a wild-type J gene carried on a plasmid that could provide J protein. An analysis of DNA synthesis during insJ mutant infection under non-permissive conditions confirmed that the J protein is not required for viral DNA synthesis.     

Effect of thedpy-20 androl-6 cotransformation markers on α-tubulin gene expression inC. elegans transformants

An -1 tubulin::lacZ fusion gene was introduced into the germline ofCaenorhabditis elegans, using eitherrol-6 ordpy-20 genomic DNA as a cotransformation marker. Distinct patterns in cellular specificity of the -1 tubulin::lacZ fusion gene expression were observed, depending on the cotransformation marker used. For therol-6 marker, the tubulin fusion gene was expressed in several neurons in the head and tail ganglia and a set of 38–39 ventral cord motor neurons along the body length of the animal during larval and adult development. In contrast, for thedpy-20 marker system, not only were fewer neurons stained in the head and tail region, but also the staining of ventral cord motor neurons was extremely reduced both in number and intensity. Thedpy-20 marked-mediated suppression of the -1 tubulin gene expression was observed both in thecis andtrans configurations. Similar down-regulation in the ventral cord motor neurons was observed when the -2 tubulin::lacZ fusion gene construct was tested in these experiments using thedpy-20 marker. In controls, where the tubulin fusion gene was directly injected to obtain transformants without any marker DNA, the cellular staining pattern was close to the fusion gene expression observed with therol-6 marker DNA. These results underline the importance of the choice of transformation marker system in generation of the transgenic animals, and reveal a down-regulation of the -tubulin fusion gene expression in the ventral cord motor neurons in transgenic animals when thedpy-20 gene was used as a cotransformation marker.     

Variants within the 5′-flanking regions of bovine milk-protein-encoding genes. III. Genes encoding the Ca-sensitive caseins αs1, αs2 and β

The 5-flanking regions of the Ca-sensitive casein-encoding gene family were analysed for DNA variants by automated DNA sequencing of 13 cows belonging to seven breeds. About 1 kbp of each 5-flanking region, including non-coding exon I, was amplified by PCR and sequenced bidirectionally. A total number of 34 variable sites (17 for the _s1, 10 for the _s2, and 7 for the casein encoding gene) was identified. Variants were computer-analysed for location in putative regulatory sites in order to predict potential influences on gene expression.     

Nucleotide sequence of the region encompassing the int gene of a cryptic prophage and the dna Y gene flanked by a curved DNA sequence of Escherichia coli K12

Summary The nucleotide sequence of a 2.5 kb region encompassing a curved DNA segment (BENT-9) randomly cloned from the total Escherichia coli chromosome was determined. This region was found to contain the dnaY gene encoding a transfer RNA. The curved DNA structure was demonstrated to be located just upstream of the dnaY promoter. The results of sequencing further revealed that the int gene of a cryptic prophage, qsr, which has been shown to be present in the E. coli genome, is located next to the dnaY gene.     

Use of site-specific recombination to regenerate selectable markers

Summary A method which allows the repeated use of a single selectable marker in DNA transformations was demonstrated. This marker regeneration method employed portions of the Saccharomyces cerevisiae 2 m circle plasmid: the inverted repeat sequences (FRTs), and the FLP gene whose product, a site-specific recombinase, catalyzes recombination events between FRTs. When FRTs were oriented as direct repeats and integrated into the genome of the yeast Pichia pastoris, FLP-mediated recombination resulted in the efficient and precise deletion of DNA located between the repeats. In the example described, the S. cerevisiae ARG4 gene, placed between a set of FRTs and integrated into Pichia in a prior transformation, was deleted by FLP, thereby regenerating an arginine-requiring phenotype in the P. pastoris strain.     

Cloning and expression of the SalI restriction-modification genes of Streptomyces albus G

Summary The Streptomyces albus G genes (salR and salM) for the class II restriction enzyme SalI (SalGI) and its cognate modification enzyme were cloned in Streptomyces lividans 66. Selection was initially for the salR gene. From a library of S. albus G DNA in the high copy number plasmid pIJ486 several clones of S. lividans were obtained that were resistant to phage C31 unmodified at the many SalI sites in its DNA, but were sensitive to modified phages last propagated on a restriction-deficient, modification-proficient mutant of S. albus G. SalI activity was detected in cell-free extracts of the clones, though only at levels comparable with that in S. albus G. Five different recombinant plasmids were isolated, with inserts of 5.6, 5.7, 8.9, 10 and 18.9 kb that contained a common region of 4.5 kb. These plasmids could not be digested by SalI, although the vector has four recognition sites for this enzyme, indicating that the salM gene was also cloned and expressed. Subcloning experiments in S. lividans indicated the approximate location of salR and salM, and in Escherichia coli led to detectable expression of salM but not of salR. A variety of previously isolated S. albus G mutants affected in aspects of SalI-specific restriction and modification were complemented by the cloned DNA; they included a mutant temperature-sensitive for growth apparently because of a mutation in salM. Southern blotting showed that DNA homologous to the cloned sal genes was present in Xanthomonas and Rhodococcus strains, but not detectably in Herpetosiphon strains, all of which produce SalI isoschizomers.     

Cloning of Candida pelliculosa beta-glucosidase gene and its expression in Saccharomyces cerevisiae

Summary Candida pelliculosa var. acetaetherius is a strain of yeast which can utilize cellobiose as the carbon source. From a gene library prepared from this yeast, the -glucosidase gene has been cloned in a S. cerevisiae host using a chromogenic substrate, 5-bromo-4-chloro-3-indolyl--glucoside as an indicator. It was proved by Southern analysis that the DNA fragment carrying the -glucosidase gene originated from C. pelliculosa. -Glucosidase produced by S. cerevisiae transformants was secreted into the periplasmic space. In Candida, -glucosidase was not induced by cellobiose but was derepressed by lowering the concentration of glucose. The regulation of -glucosidase synthesis in S. cerevisiae carrying the cloned -glucosidase was not clear compared with that in Candida, however, the enzyme activity in low glucose medium (0.05%) was reproducibly higher than in high glucose medium (2%). We have found the sequence that controls the expression of the -glucosidase gene negatively in S. cerevisiae.     

Construction of an amylolytic industrial strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis α-amylase gene

The gene encoding Schwanniomyces occidentalis -amylase (AMY) was introduced into the chromosomal sequences of an industrial strain of Saccharomyces cerevisiae. To obtain a strain suitable for commercial use, an -integrative cassette devoid of bacterial DNA sequences was constructed that contains the AMY gene and aureobasidin A resistance gene (AUR1-C) as the selection marker. The AMY gene was expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p). The -amylase activity of Sacc. cerevisiae transformed with this integrative cassette was 6 times higher than that of Sch. occidentalis. The transformants (integrants) were mitotically stable after 100 generations in nonselective medium.     

Agrobacterium-mediated production of transgenic rice plants expressing a chimeric α-amylase promoter/β-glucuronidase gene

We have successfully transferred and expressed a reporter gene driven by an -amylase promoter in a japonica type of rice (Oryza sativa L. cv. Tainung 62) using the Agrobacterium-mediated gene transfer system. Immature rice embryos (10–12 days after anthesis) were infected with an Agrobacterium strain carrying a plasmid containing chimeric genes of -glucuronidase (uidA) and neomycin phosphotransferase (nptII). Co-incubation of potato suspension culture (PSC) with the Agrobacterium inoculum significantly improved the transformation efficiency of rice. The uidA and nptII genes, which are under the control of promoters of a rice -amylase gene (Amy8) and Agrobacterium nopaline synthase gene (nos), respectively, were both expressed in G418-resistant calli and transgenic plants. Integration of foreign genes into the genomes of transgenic plants was confirmed by Southern blot analysis. Histochemical localization of GUS activity in one transgenic plant (R0) revealed that the rice -amylase promoter functions in all cell types of the mature leaves, stems, sheaths and roots, but not in the very young leaves. This transgenic plant grew more slowly and produced less seeds than the wild-type plant, but its R1 and R2 progenies grew normally and produced as much seeds as the wild-type plant. Inheritance of foreign genes to the progenies was also confirmed by Southern blot analysis. These data demonstrate successful gene transfer and sexual inheritance of the chimeric genes.     

Temporally uncontrolled expression of linearized plasmid DNA which carries bacterial chloramphenicol acetyltransferase gene withXenopus cardiacα-actin promoter after injection intoXenopus fertilized eggs

Summary Circular and linearized plasmid DNA which contained bacterial chloramphenicol acetyltransferase (CAT) gene connected toXenopus cardiac-actin promoter was injected intoXenopus fertilized eggs to study their expression in the course of early embryonic development. While circular DNA was slightly replicated and expressed only after embryos reached neurula stage, linearized DNA formed a large amount of concatemers, and was expressed as early as at blastula stage, or about 14 hr earlier than the time of circular DNA expression. Similarly earlier expression of linearized DNA occurred slightly more strongly when the DNA was injected into presumptive dorsal than in ventral blastomeres at 4-cell stage, and the expression was not affected when embryos were dissociated at blastula stage and their cells were cultured under reaggregating or nonreaggregating conditions. These results show that although circular actin-CAT fusion gene is expressed during development according to endogenous temporal control, the expression of linearized DNA deviates from such developmental control even though it contains intact promoter of-actin gene. It is then recommended that study of the control of the expression of exogenously-introduced DNA inXenopus fertilized eggs should be carried out with circular but not linearized plasmids.     

Sequence variation at theSec-1 locus of rye,Secale cereale (Poaceae)

This paper describes the structure of a 9.2-kb repeat unit of DNA, which represents one-secalin gene and spacer sequence located at theSec-1 locus on the short arm of chromosome 1 of rye. The gene units at theSec-1 locus comprise 1.1 kb representing the gene and 8.1 kb of spacer sequence separating the genes. A sequence comparison of nine genes and their promoter regions from theSec-1 locus, reveals that there is greater variation within the coding sequence than there is within the promoter regions. The gene sequence variation is discussed in terms of the size variation seen for the-secalin proteins in rye species. The results include a comparison of promoter sequences from members of the Triticeae to examine the degree of conservation between other seed storage protein genes.