Expression of heterologous genes for wall teichoic acid in Bacillus subtilis 168

Summary A localized region of low DNA sequence homology was revealed in two strains of Bacillus subtilis by a specific 100-fold reduction in transformation by W23 DNA of the tag1 locus, a teichoic acid marker of strain 168. Fifty nine rare recombinants, hybrid at this locus, had all acquired donor-specific phage resistance characters, while losing those specific to the 168 recipient. Chemical analysis of isolated cell walls showed that these modifications are associated with major changes in the wall teichoic acids. Genetic analysis demonstrated that determinants for the ribitol phosphate polymer of strain W23 had been transferred to 168, replacing those for the glycerol phosphate polymer in the recipient. All W23 genes coding for poly(ribitol phosphate) in the hybrids and those specifying anionic wall polymers in strain 168 are clustered near hisA. In addition to tag1, the region exchanged extends just beyond gtaA in some hybrids, whereas in others it may include the more distant gtaB marker, encompassing a region sufficient to contain at least 20 average-sized genes. Surface growth, flagellation, transformability and sporulation all appeared normal in hybrids examined. Recombinants without a major wall teichoic acid from either strain were not found, suggesting that an integral transfer of genes for poly(ribitol phosphate) from W23 had occurred in all hybrids isolated. We interpret these results as indicating an essential role for anionic wall polymers in the growth of B. subtillis.     

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.     

In addition to the ARS core, the ARS box is necessary for autonomously replicating sequences

Summary Autonomously replicating sequences (ARSs) were cloned from nuclear and mitochondrial DNA of D. melanogaster using YIp5, which is composed of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^- yeast strain as the recipient. The nucleotide sequences of six ARSs, two from nuclear bulk, two from the nuclear 1.688 satellite, and two from mitochondorial DNA, were determined. The relationship between the transformation frequency and the inclusion of the ARS core, 5 _T ^A TT-TAT _A ^G TTT _T ^A 3, of these fragments was analysed. All the ARSs contained an ARS core or a single base change of it. However, not all the fragments that contained a single base change of the ARS core were able to transform the recipient cells, suggesting that certain bases in the ARS core were not exchangeable. It is suggested by transformation experiments with subfragments that in addition to an ARS core, an ARS box which is located within 25 bp upstream of the ARS core and whose sequence is composed of 5TNT _G ^A AA 3, is necessary for autonomous replication.     

Mutations that increase the mitotic stability of minichromosomes in yeast: characterization of RAR1

Summary In an attempt to identify proteins involved in the initiation of DNA replication, we have isolated a series of Saccharomyces cerevisiae mutants in which the function of putative replication origins is affected. The phenotype of these Rar^- (regulation of autonomous replication) mutants is to increase the mitotic stability of plasmids whose replication is dependent on weak ARS elements. These mutations are generally recessive and complementation analysis shows that mutations in several genes may improve the ability of weak ARS elements to function. One mutation (rar1-1) also confers temperature-sensitive growth, and thus an essential gene is affected. We have determined the DNA sequence of the RAR1 gene, which reveals an open reading frame for a 48.5 kDa protein. The RAR1 gene is linked to rna1 on chromosome XIII.     

Sequence of the mglB gene from Escherichia coli K12: comparison of wild-type and mutant galactose chemoreceptors

Summary The mglB gene of Escherichia coli codes for a galactose-binding protein (GBP) that serves both as the galactose chemoreceptor and as the recognition component of the -methylgalactoside transport system. The mglB551 mutation eliminates the chemotactic function of GBP without altering its transport or substrate-binding properties. To investigate the interaction between GBP and Trg, the chemotactic signal transducer for galactose, we sequenced the mglB genes from wild-type and mglB551 mutant strains. The mutation causes the replacement of Gly₇₄ of GBP by Asp. This residue is located in alpha-Helix III at the tip of the P domain in the GBP tertiary structure farthest removed from the substrate-binding cleft between the P and Q domains. We conclude that Helix III must be part of, or at least adjacent to, the recognition site for Trg. Our sequence also included part of the mglA gene, which is immediately distal to mglB. The amino acid sequence deduced for the beginning of the MglA protein showed homology with a family of polypeptides that contain an ATP-binding site and are components of binding-protein-dependent transport systems.     

Restriction pattern analysis of deoxyribonucleic acid isolated from callus and cell suspension of actinorhizal and non-actinorhizal Betulaceae

Using cell suspensions, a method was elaborated to isolate high-molecular-weight genomic deoxyribonucleic acid (DNA; 65 MDa or more) from members of the Betulaceae: Alnus incana (L.) Moench, Alnus glutinosa (L.) Gaertn. and Betula papyrifera Marsh. The method was also effective for isolation of DNA from callus cells. Based on the chemical lysis of protoplasts, this procedure yielded 130 μg (callus) to 250 μg (cell suspension) of DNA (g fresh cells)⁻¹, with a ratio A₂₀₀/A₂₈ of 1.7–2.0. The purified DNA obtained, formed distinct bands when restricted fragments were electrophoresed. Among the 10 endonucleases used for restriction analysis of Alnus glutinosa, Alnus incana and Betula papyrifera genomes, PvuI1 (EC 3.1.23.33) was unique in giving identical patterns for the two Ainus species. An unusual pattern occurred when Al-2 DNA was restricted with Ava II (EC 3.1.23.4). It formed a ladder with a repeating fragment unit of 181 base pairs long. With the enzymes tested, no differences in restriction patterns were observed among clones of Alnus incana (AI-2 vs AI-2), Betula papyrifera (BP-4 vs BP-8) and subclones of Ainus glutinosa AG-1 (PLFJ709 vs LF1709), suggesting genetic stability of the Betulaceae cultures.     

Molecular cloning of the plant plasma membrane H+-ATPase

Summary Mineral transport across the plasma membrane of plant cells is controlled by an electrochemical gradient of protons. This gradient is generated by an ATP-consuming enzyme in the membrane known as a proton pump, or H⁺-ATPase. The protein has a catalytic subunit of Mr=100,000 and is a prominent band when plasma membrane proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We generated specific rabbit polyclonal antibody against the Mr=100,000 H⁺-ATPase and used the antibody to screen λgtll expression vector libraries of plant DNA. Several phage clones producing immunoreactive protein, and presumably containing DNA sequences for the ATPase structural gene, were isolated and purified from a carrot cDNA library and a Arabidopsis genomic DNA library. These studies represent our first efforts at cloning the structural gene for a plant plasma membrane transport protein. Applicability of the technique to other transport protein genes and the potential for use of recombinant DNA technology in plant mineral transport research are discussed.     

Improved methods of estimating root length using a photocopier,a light box and a bar code reader

Photocopying was found to be a rapid method of making a permanent record of a root sample. The method used produced a copy with white roots against a black background. Manual estimates of root length were made from photocopies using a light box. The number of intersections visible when laid over a copy of a white on black regular square grid was counted. Automated estimates of root length were made by scanning a photocopy with a bar code reader in place of a pen in a computer-driven graph plotter. Roots >0.2 mm diameter were resolved with precision and speed.