Showing papers in "Molecular Genetics and Genomics in 1984"

A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance.

Abstract: Mutations at the URA3 locus of Saccharomyces cerevisiae can be obtained by a positive selection. Wild-type strains of yeast (or ura3 mutant strains containing a plasmid-borne URA3+ gene) are unable to grow on medium containing the pyrimidine analog 5-fluoro-orotic acid, whereas ura3- mutants grow normally. This selection, based on the loss of orotidine-5'-phosphate decarboxylase activity seems applicable to a variety of eucaryotic and procaryotic cells.

Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations.

Abstract: A gene bank of Sau3A partially digested Candida albicans DNA in vector YEp13 was used to complement a ura3 mutation (orotidine-5'-phosphate decarboxylase, OMPdecase) in S. cerevisiae. Two plasmids which complemented ura3 and showed clear linkage of Ura+ and plasmid markers were selected for further study. Both plasmids also complemented the corresponding OMPdecase mutation (pyrF) in E. coli. Restriction mapping and subcloning studies localized the OMPdecase complementing activity to a region common to both plasmids. Probes prepared from this common region hybridized specifically to C. albicans DNA and not to E. coli or S. cerevisiae DNA. Southern blot analysis also showed that the restriction map of the ura3 complementing region of one plasmid was colinear with C. albicans genomic DNA. Expression of the OMPdecase complementing gene in E. coli and S. cerevisiae was not dependent upon orientation relative to vector sequences, suggesting that promotion could be occurring within the C. albicans fragment. Expression was sufficient to allow complementation in S. cerevisiae with integrating as well as high copy number vectors.

High frequency mobilization of gram-negative bacterial replicons by the in vitro constructed Tn5-Mob transposon.

Abstract: A DNA fragment of the broad host range plasmid RP4 carrying the cis-acting DNA recognition site for conjugative DNA transfer between bacterial cells (Mobsite) was cloned into the kanamycin-neomycin resistance transposon Tn5. Using conventrional transposon mutagenesis techniques the new transposon, called Tn5-Mob, can easily be inserted into the host DNA of gram-negative bacteria. A host replicon carrying Tn5-Mob is then mobilizable into any other gram-negative species if the transfer functions of plasmid RP4 are provided in trans. The potential of Tn5-Mob was demonstrated by mobilizing Rhizobium meliloti plasmids as well as the E. coli chromosome at high frequencies.

The DNA of Arabidopsis thaliana

Abstract: Arabidopsis thaliana is a small flowering plant of the mustard family. It has a four to five week generation time, can be self- or cross-pollinated and bears as many as 104 seeds per plant. Many visible and biochemical mutations exist and have been mapped by recombination to one of the five chromosomes that comprise the haploid karyotype. With the experiments reported here we demonstrate that Arabidopsis has an extraordinarily small haploid genome size (approximately 7×107 nucleotide pairs) and a low level of cytosine methylation for an angiosperm. In addition, it appears to have little repetitive DNA in its nuclear DNA, in contrast to other higher plants.

Physical and genetic analysis of a symbiotic region of Rhizobium meliloti: Identification of nodulation genes

Abstract: A 135 kb long segment of the symbiotic region of the Rhizobium meliloti megaplasmid was mapped with the help of a Rhizobium meliloti gene library, made in the cosmid vehicle pJB8. A set of overlapping cosmid clones was used to identify the inserts in R-primes carrying megaplasmid sections, and to map 20 deletion mutations and 24 insertion mutations with Nod- or Fix- phenotypes. This led to the identification of DNA regions carrying nod or fix (nif) genes. The results of this study correlate well with transcription data of nodule-specific expression of plasmid sequences. The nod mutations were localized in two groups. Using directed Tn5 mutagenesis, correlated physical-genetic maps for these regions were established. One nod gene cluster is about 2.5–3.0 kb in size and carries genes involved in root hair curling, a very early step in nodule formation. Mutations in these genes can be complemented by sym plasmids of other Rhizobium species, such as Rhizobium leguminosarum. We designate these genes as “common” nod genes because mutations in them can be complemented by plasmids derived from different Rhizobium strains. The other nod gene cluster consists of a 2 kb and a 1 kb long DNA segment, separated by a 1 kb region nonessential for nodulation. These nod genes are probably involved in the host specificity of nodulation.

A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjacent to Tn917 insertions.

Abstract: A rapid and general procedure has been devised for the pBR322-mediated cloning in Escherichia coli of Bacillus subtilis chromosomal DNA extending in a specified direction from any Tn917 insertion. Derivatives of Tn917 have been constructed that contain a pBR322-derived replicon, together with a chloramphenicol-resistance (Cmr) gene of Gram-positive origin (selectable in B. subtilis), inserted by ligation in two orientations into a SalI restriction site located near the center of the transposon. When linearized plasmid DNA carrying such derivatives was used to transform to Cmr B. subtilis bacteria already containing a chromosomal insertion of Tn917, the pBR322 sequences efficiently became integrated into the chromosomal copy of the transposon by homologous recombination. It was then possible to clone chromosomal sequences adjacent to either transposon insertion junction into E. coli, using a selection for ampicillin-resistance, by transforming CaCl2-treated cells with small amounts of insert-containing DNA that had been digested with various restriction enzymes and then ligated at a dilute concentration. Because pBR322 sequences may be inserted by recombination in either orientation with respect to the transposon arms, a single restriction enzyme (such as EcoRI or SphI) that has a unique recognition site in pBR322 DNA may be used to separately clone chromosomal DNA extending in either direction from the site of any transposon insertion. A family of clones generated from the region of an insertional spo mutation (spoIIH::Tn917) was used in Southern hybridization experiments to verify that cloned material isolated with this procedure accurately reflected the arrangement of sequences present in the chromosome. Strategies are discussed for taking advantage of certain properties inherent in the structure of clones generated in this way to facilitate the identification and study of promoters of insertionally mutated genes.

Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12: identification of a new mutation (recQ1) that blocks the RecF recombination pathway.

Abstract: An Escherichia coli K12 mutant resistant to thymineless death (TLD) was isolated, and its genetic analysis led us to identify a new mutation (recQ1) located between corA and metE on the standard linkage map. The mutation was found to result in increased sensitivity to ultraviolet light and deficiency in conjugational recombination when placed in the recBC sbcB background, indicating that it blocked the RecF pathway of recobbination. It seemed likely that this mutation is also capable of causing partial resistance to TLD, but we reserve the possibility of a separate mutation closely linked to recQ1 giving rise to this phenotype. The original mutant was shown to carry an additional mutation probably in the vicinity of the uhp locus, which was also required for the full TLD resistance of the mutant to be expressed.

Genetic analysis of the individual T-DNA genes of Agrobacterium tumefaciens ; further evidence that two genes are involved in indole-3-acetic acid synthesis

Abstract: The T-DNA genes of Ti plasmids of Agrobacterium tumefaciens can induce tumorous growth on a wide range of dicotyledonous plants. We subcloned the individual onc genes of the pTiC58 T-DNA and reintroduced them in the T-region of the Ti plasmid gene vector pGV3850 (from which the onc genes had been removed (Zambryski et al. 1983)). These experiments were designed to analyze the contribution of each onc gene to the development of a tumor and have fulfilled two purposes. First, it was found that only the strains carrying gene 4 produced tumors without the aid of other T-DNA genes; in cell culture these tumors sprout shoots. Second, the shoot-forming phenotype of tumors induced by agrobacteria carrying Ti plasmids defective in either gene 1 or gene 2 can be restored to wildtype phenotype by simple coinfection with Agrobacterium strains whose Ti plasmids contain respectively only gene 2, or only gene 1 in their T-region. A parallel experiment demonstrated that the combined action of genes 1 and 2 is sufficient to induce tumor formation on tobacco plantlets.

Cloning of the repressor protein gene of iron-regulated systems in Escherichia coli K12.

Abstract: In Escherichia coli the iron uptake systems are regulated by the fur gene product. The synthesis of the outer membrane proteins fiu, fepA, fecA, fhuA, fhuE and cir is derepressed at low iron concentrations in the medium or constitutive in a fur mutant. The fur gene region cloned into pACYC184 was analysed by restriction analysis, Tn1000 mutagenesis and complementation studies. The presence of fur+ plasmids repressed synthesis of the proteins fepA, fecA, fhuE and cir in a chromosomal fur mutant. More quantitatively, the repression to wild-type levels was shown with lac fusions to the genes fiu, fepA and cir. In minicells an 18,000 dalton protein was identified as the fur gene product. Correlated with the fur protein a slightly smaller protein, possibly a degradation product, was observed. The gene fur was mapped on the E. coli chromosome near nagA at about 15.5 min.

Observations on the formation of clones containing araB-lacZ cistron fusions.

Abstract: Casadaban (1976) developed a technique for isolating E. coli clones containing fusions of the amino terminal-encoding portion of any cistron with the carboxy terminal-encoding portion of lacZ. The technique utilizes prophage Mu homology to bring the two cistrons into proximity. I have followed the appearance over time of colonies containing araB-lacZ fusions from a strain where the begining of the araB cistron is connected to lacZ by an intact Mucts62 prophage. Cultures of the starting strain grown on a variety of media have fewer than 2 in 1010 cells capable of forming colonies within three days after plating on selective arabinose-lactose medium. At 32°C, there is a delay of between 4 and 19 days before the first colony appears. The kinetics of colony appearance over the next two to four weeks then shows a rapid increase in the number of new colonies emerging per day followed by a decline. The pattern of colonial emergence and the final numbers of fusion colonies obtained are not grossly affected by reducing the number of cells plated over five orders of magnitude. Fusion colonies sometimes show a clustered pattern when they first emerge. Innoculation of pre-existing fusion clones at specific locations on the arabinose-lacteredselection plates seeded with the starting strain leads to the formation of inhibitory zones where no fusion colonies appear. Selection plates contain many microcolonies and papillae which do not proliferate into scoreable colonies but nonetheless contain cells capable of growth when replated on the same selective medium. Up to 39% of all plated cells are capable of producing fusion clones. The kinetics of fusion colony appearance can be altered by environmental and genetic manipulations. Partial derepression of the Mucts prophage at 37° accelerates the appearance of colonies but also reduces the final yield. Addition of limiting concentrations of glucose to the selective medium also accelerates the appearance of colonies in a specific fashion: enrichments below the level required for maximum acceleration produce a biphasic kinetics with two waves of fusion clone emergence separated by an eight-day interval. Infection with Muc + pAp phage produces dilysogens that have almost completely lost the ability to produce fusions. Infection with MuctsAampAP phage produces strains that are reduced in phage production and have delayed kinetics of fusion clone emergence. The implications of these observations for theories of hereditary change in bacteria are discussed.

Tn4001: a gentamicin and kanamycin resistance transposon in Staphylococcus aureus.

Abstract: We describe a 4.5 kilobase transposon, Tn4001, which mediates resistance to gentamicin, tobramycin and kanamycin in Staphylococcus aureus. Originally detected in plasmid pSK1, Tn4001 was shown to undergo rec-independent transposition to the chromosome from this plasmid and from an inserted derivative of the plasmid pII147. Heteroduplexes between plasmids with and without Tn4001 demonstrated a characteristic stem and loop structure with inverted repeats of approx. 1.3 kilobases.

Effect of ruv mutations on recombination and DNA repair in Escherichia coli K12.

Abstract: Mutation of the ruv gene of E. coli is associated with sensitivity to radiation, and filamentous growth after transient inhibition of DNA synthesis. The filamentation of ruv strains is abolished by mutations in sfiA or sfiB that prevent SOS induced inhibition of cell division, but this does not restore resistance to UV radiation. Double mutants carrying both ruv and uvr mutations are considerably more sensitive to UV radiation than the single mutants, but there is no additive effect of ruv with recA, recF, recB, or recC mutations. ruv mutations have little effect on conjugal recombination in wild-type strains but confer recombination deficiency and extreme sensitivity to ionizing radiation in recBC sbcB strain. These results, together with the fact that ruv strains are excision proficient and mutable by UV light, are interpreted to suggest that the ruv + product is involved in recombinational repair of damaged DNA rather than in cell division as suggested by Otsuji et al. (1974).

Repair of DNA double-strand breaks in Escherichia coli K12 requires a functional recN product.

Abstract: Mutation of the recN gene of Escherichia coli in a recBC sbcB genetic background blocks conjugational recombination and confers increased sensitivity to UV light and mitomycin C. The basis for this phenotype was investigated by monitoring the properties associated with recN mutations in otherwise wild-type strains. It was established that recN single mutants are almost fully resistant to UV irradiation, and that there is no detectable defect in repair of UV lesions by excision, error-prone, or recombinational mechanisms. However, recN mutations confer sensitivity to mitomycin C and ionizing radiation both in wild-type and recB sbcB strains. The sensitivity to ionizing radiation is correlated with a deficiency in the capacity to repair DNA double-strand breaks by a UV inducible mechanism. Recombinant λ phages that complement the recombination and repair defects of recN recBC sbcB mutants have been identified, and the recN gene has been cloned from these phages into a low copy-number plasmid.

The DNA sequnce of the transposable element Ac of Zea mays L.

Abstract: The sequence of the Ac element isolated from the wx-m7 allele has been determined. The Ac element is 4563 bp long. A central portion of roughly 3.1 kb is occupied by three open reading frames, two of which point in one direction and the third in the opposite direction. One of the reading frames potentially encodes a protein with a ten-fold repeat of pro gluN and pro glu dipeptides near its N-terminus. The sequences outside the open reading frames are characterized by the presence of a number of direct and inverted repeats. The Ac element may thus have evolved from a simpler progenitor structure. The sequence we have determined for the Ac from the wx-m7 allele differs in a few key positions from that reported for the Ac element from the wx-m9 allele (Pohlman et al. 1984). We have resequenced these positions in both Ac elements and find them to be identical. We conclude that the phenotypic differences between the two waxy alleles are not caused by structural differences in the Ac elements but rather may be attributable to the differences in their insertion sites.

Behaviour of the transposons Tn5 and Tn7 in Xanthomonas campestris pv. campestris

Abstract: Xanthomonas campestris pv. campestris was tested for its ability to maintain various plasmids after they had been transferred by conjugation from Escherichia coli donors. Broad host-range plasmids belonging to incompatibility groups P and Q could be maintained but X. campestris was unable to support replication of narrow host-range ColE1, pACYC184 and pBR325 replicons. Delivery systems based on E. coli donors of suicide plasmids and on X. campestris Hfrs were used to introduce Tn7 and Tn5 into X. campestris. Tn7 insertions were recovered at high frequency while Tn5 transposed at low frequency. Three auxotrophic Tn5 insertions were isolated but transposition of Tn7 into the X. campestris genome did not generate any auxotrophs. DNA hybridization analysis showed that Tn7 had inserted into the same “hot spot(s)” in all cases tested.

Nucleotide sequence of the ribulosebisphosphate carboxylase gene from Rhodospirillum rubrum

Abstract: The nucleotide sequence of a cloned DNA fragment encoding ribulose bisphosphate carboxylase from the purple non-sulfur bacterium Rhodospirillum rubrum has been determined. The deduced amino acid sequence of a 1398 nucleotide open reading frame exhibits weak overall homology to the sequences reported for analogous enzymes from cyanobacteria, algae and angiosperms. Thus knowledge of the sequence is useful in attempts to identify structural features of the enzyme which are essential to catalysis. The gene is flanked by nucleotide sequences similar to those implicated in the initiation of translation and termination of transcription in other bacteria.

Localized Mutagenesis in Rhizobium japonicum

Abstract: The slow-growing soybean symbiont, Rhizobium japonicum, has not readily been accessible so far to classical mutational analysis of genes responsible for symbiotic nitrogen fixation. We have overcome part of this problem by the successful application of a site-directed mutagenesis technique to this organism. The following steps are involved: (i) local Tn5 mutagenesis, in E. coli, of cloned R. japonicum DNA (e.g. the nifDK operon); (ii) conjugational transfer of the mutated DNA into R. japonicum using vectors which are unable to replicate there; (iii) selection of R. japonicum exconjugants which have exchanged their wild-type genomic DNA region for the Tn5-containing fragment by homologous recombination. While using this technique it appeared mandatory to distinguish double-crossover-events (true replacements) from single-crossover events (replicon fusions or cointegrations). Only the true replacement mutants were genetically stable; their phenotypes were determined with respect to nodulation (Nod) and nitrogen fixation (Fix) by plant infection tests. Tn5 mutations within nifD and nifK caused a Nod+ Fix- phenotype, whereas mutants with insertions in the immediate vicinity on either side of nifDK were found to be Nod+ Fix+, suggesting that genes flanking nifDK may not be involved in the nitrogen fixing symbiosis. Nodule reisolates were found to carry Tn5 at their original locations.

The pAtC58 plasmid of Agrobacterium tumefaciens is not essential for tumour induction

Abstract: When the 225 kilobase (kb) cryptic plasmid of Rhizobium meliloti 41 is introduced into Agrobacterium tumefaciens C58, the resident plasmid pAtC58 (410 kb) is lost, probably because of incompatibility. The strain of A. tumefaciens cured of pAtC58 is still oncogenic, showing that pAtC58 does not control functions essential for tumour formation in the tomato and in Kalanchoe daigremontiana.

Kinetic impairment of restrictive streptomycin-resistant ribosomes.

Abstract: Comparisons in vivo and in vitro of wild-type and otherwise isogenic bacteria with five different mutant alleles of the gene (rpsL) specifying ribosomal protein S12, all resistant to high levels of streptomycin, show that the streptomycin-resistant (Smr) phenotype can be subdivided into major groups: restrictive and non-restrictive. The restrictive bacteria have a characteristically lower frequency of nonsense suppression in vivo, and are also slower than the wild type in their rate of protein synthesis. Non-restrictive Smr bacteria on the other hand do not differ significantly from the wild type either in nonsense suppression frequencies or in the rate of translation.

Changes in T-DNA methylation and expression are associated with phenotypic variation and plant regeneration in a crown gall tumor line.

Abstract: Phenotypic variation of an octopine-type crown gall tumor line resulting from changes in the pattern of T-DNA methylation and expression is described. Variants that grow as unorganized callus always express T-DNA transcripts 1 and 2. In shoot-forming variants (teratomas) only T-DNA transcript 4 is expressed. This line also regenerates normal-appearing, rooted plants in which all T-DNA expression is suppressed. Tissues from these plants require phytohormones for growth in vitro. These plants are selffertile and transmit T-DNA through meiosis, and T-DNA suppression is maintained in the next generation. After treatment of regenerated plant tissue with 5-azacytidine, an inhibitor of DNA methylation, T-DNA transcription and phytohormone-independent tumorous growth resume. The T-DNA of cell lines in which T-DNA is not expressed is highly methylated, whereas the level of T-DNA methylation is reduced in 5-azacytidine treated cells that resume T-DNA expression and phytohormone-independent growth. The correlation between the degree of T-DNA methylation and the level of T-DNA expression indicates that hypermethylation is responsible for the suppression of T-DNA transcription.

Cloning and expression of the Rhodospirillum rubrum ribulosebisphosphate carboxylase gene in E. coli

Abstract: A genomic library of Rhodospirillum rubrum DNA was constructed in the phage replacement vector λ1059. Recombinant phage carrying the gene for ribulose 1,5-bisphosphate (RuBP) carboxylase were identified by radioimmune blotting of plaques. A 6.6 kb EcoRI fragment from one of the immunologically positive phage was subcloned into pBR325 and a plasmid which conferred low levels of enzyme activity in E. coli was recovered. Expression of the gene was dependent upon the orientation of the restriction fragment in pBR325, suggesting that transcription originated on a plasmid promoter. In order to increase expression, a new plasmid was constructed by replacing the tetracycline resistance determinant of pBR322 with a restriction fragment from phage M13mp7 which encoded part of the lac operon. A 2.4 kd restriction fragment containing the RuBP carboxylase gene was then cloned into the unique BamHI site of the lac DNA. Induction of lac-transcription resulted in a high level of expression of a fully functional RuBP carboxylase enzyme, which was purified to homogeneity. Southern hybridization analysis of the R. rubrum genome with a restriction fragment encoding part of the enzyme indicated only one copy of the gene.

Restoration of virulence of Vir region mutants of Agrobacterium tumefaciens strain B6S3 by coinfection with normal and mutant Agrobacterium strains

Abstract: Three avirulent Tn7 insertion mutants mapping in the vir E region of the Agrobacterium tumefaciens plasmid pTiB6S3 regain virulence by co-infection with several wildtype strains and with a number of strains carrying mutations in other regions of the Ti plasmid. This finding indicates that during tumour induction normal Agrobacterium strains produce a diffusable factor required for transformation and might allow the isolation of such a factor.

A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for some tRNA nonsense suppressors.

Abstract: A spontaneous mutant was isolated that harbors a weak suppressing activity towards a UAG mutation, together with an inability to grow at 43° C in rich medium. The mutation is shown to be associated with an increased misreading of UAG at certain codon contexts and UAA. UGA, missense or frameshift mutations do not appear to be misread to a similar extent. The mutation gives an increased efficiency to several amber tRNA suppressors with-out increasing their ambiguity towards UAA. The ochre suppressors SuB and Su5 are stimulated in their reading of both UAG and UAA with preference for UAG. An opal suppressor is not affected. The effect of the mutation on the efficiency of amber and ochre suppressors is dependent on the codon context of the nonsense codon.

Identification of a Ty insertion within the coding sequence of the S. cerevisiae URA3 gene.

Abstract: Insertion mutations in yeast caused by the transposable element Ty have been identified at several genetic loci. In all cases so far, the site of Ty insertion has been in the 5′ non-coding region of the affected gene. Experiments presented here demonstrate that the ura3-52 mutation, a non-reverting ura3 mutation, is caused by a Ty insertion mutation within the coding region of the URA3 gene. This Ty insertion within a gene has a similar structure to those in non-coding regions.

Genetics of mitochondria in Drosophila: mtDNA inheritance in heteroplasmic strains of D. mauritiana

Abstract: The transmission rules of the mitochondrial genome have been established from the analysis of nucleomorph segregation in several Drosophila mauritiana heteroplasmic strains. The mode of segregation is independent of the nature and the initial frequencies of the nucleomorphs. As predicted from a genetic drift model, the quasicomplete sorting-out will need as many as 500 generations. The number of segregating units, estimated at about 400, fits well the number of mitochondria observed in animal cells.

Plasmids in different strains of Streptomyces ambofaciens: free and integrated form of plasmid pSAM2.

Abstract: Five strains of Streptomyces ambofaciens were examined for their plasmid content. Among these strains, four belong to the same lineage (strains B) and the other was isolated independently (strain A). A large plasmid (ca. 80 kb), called pSAM1 in this paper and already described, was present in all B strains, and absent in strain A. A second plasmid, not described before, was found as covalently closed circular DNA in two of the four B strains. This plasmid with a size 11.1 kb was called pSAM2. A restriction map for 14 enzymes was established. Hybridization experiments showed that a unique sequence homologous to this plasmid is integrated in a larger replicon, which is not pSAM1 and is probably the chromosome, in all B strains and not in strain A. It seems probable that the integrated se1uence is the origin of the free plasmid found in two strains of the B family. It is noteworthy that the integrated form and the free plasmid may be found together. Transformation experiments proved that pSAM2 may be maintained autonomously in S. ambofaciens strain A and in S. lividans. pSAM2 is a self-transmissible plasmid, able to elicit the lethal zygosis reaction. pSAM2 was compared to the plasmids SLP1, pIJ110 and pIJ408, which all come from integrated sequences in three Streptomyces species and are found as autonomous plasmids after transfer to S. lividans. If pSAM2 resembles these plasmids in its origin, it does not appear to be related directly to them. Concerning their plasmid content, the two isolates of S. ambofaciens are very different. One of them contains neither pSAM1 not pSAM2. As this isolate produces spiramycin, these plasmids probably do not play an important role in spiramycin production. Apart from its intrinsic biological interest, pSAM2 may be useful in the construction of cloning vectors for S. ambofaciens. Very stable transformants might be obtained in certain strains of S. ambofaciens, because of the possibility of integration of the pSAM2 derivative vector.

DNA amplification and an unstable arginine gene in Streptomyces lividans 66

Abstract: Streptomyces lividans 66 produced spontaneous chloramphenicol-sensitive mutants (Cmls) at a frequency of about 1% of spores. The Cmls mutant strains were very unstable, giving Arg- mutants at frequencies of about 25% of spores. All the Arg- mutants had amplified a particular 5.75 kb DNA fragment into tandem repeats of 250–500 copies per chromosome.

Function of RNase H in DNA replication revealed by RNase H defective mutants of Escherichia coli.

Abstract: Escherichia coli rnh mutants were isolated using localized mutagenesis and selective measurements of RNase H activity in mutagenized cell extracts with [3H]poly(rC)·poly(dG) as substrate. RNase H activity in extracts of one mutant, ON152 (rnh-91), was undetectable (less than 0.05% of that of wild-type cells). This mutant formed small colonies at 43 °C. At this temperature, accumulation of nascent fragments was more prominent in the rnh-91·polA4113 double mutant than in the polA4113 mutant; however, no accumulation was found in the rnh single mutant at 43° C. Unlike the 1–3 nucleotide primer RNA found on nascent fragments of polA4113 cells, primers from the rnh-91·polA4113 cells ranged from one to about ten bases. These results suggest that the 5′→3′ exonuclease activity of DNA polymerase I plays a major role in removal of primer RNA and that RNase H functions in an auxiliary role, excising the 5′-portion of longer primers. The rnh mutant supports replication of ColE1-type plasmids. A possible mechanism of replication of such plasmids in rnh mutants and a role of RNase H in the initiation of chromosomal replication are discussed.

The expression of heat shock genes during normal development in Drosophila melanogaster (heat shock/abundant transcripts/developmental regulation)

Abstract: Drosophila melanogaster cells and tissues respond to heat shock by dramatically altering their pattern of transcription and translation, leading to the rapid synthesis of a small number of polypeptides, the heat shock proteins (hsps). By using cloned hsp DNA we have detected sequences complementary to heat shock genes in RNA prepared from non-heat-shocked animals of different developmental stages. Hsp 83 mRNA is present at high levels in all stages examined. Hsp 68 and 70 mRNAs are present at very low levels in most stages and at slightly higher concentration in pupae. Hsp 26 and 27 mRNAs are detected in embryos. Hsp 23, 26 and 27 mRNA are barely detectable in early third instar larvae but are major components of late third instar and early pupal RNA. Hsp 22 mRNA is also detected in early pupae. Later in development the levels of the small hsp mRNAs decrease but a further peak in abundance of hsp 26 and 27 mRNAs is found in mature adult females.

Functional domains of Escherichia coli recA protein deduced from the mutational sites in the gene.

Abstract: The sites of recA mutations of Escherichia coli, recA441 (tif-1), recA1, recA430 (lexB30) and recA44, were determined by analyses of the nucleotide sequences. All mutations are single point missense mutations within the coding region of the recA gene. In the recA441, recA1, recA430 and recA44 proteins, the 38th, 160th, 204th, and 246th amino acids, respectively, from the amino terminal ends are altered. Based on the properties of these mutant proteins and modified forms of recA protein, the locations of various regions of the recA protein that are involved in binding with ATP, binding with single-stranded DNA, hydrolysis of ATP, interaction between the recA protein molecules and interaction with the lambda cI or lexA repressors are mapped on the primary structure of the protein.