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S A Lacks

Publications and source records attributed to S A Lacks.

At least 55 records · Page 3Linked to original sources
PubMed1985

Nucleotide sequence of the Dpn II DNA methylase gene of Streptococcus pneumoniae and its relationship to the dam gene of Escherichia coli.

The structural gene (dpnM) for the Dpn II DNA methylase of Streptococcus pneumoniae, which is part of the Dpn II restriction system and methylates adenine in the sequence 5'-G-A-T-C-3', was identified by subcloning fragments of a chromosomal segment from a Dpn II-producing strain in an S. pneumoniae host/vector cloning system and demonstrating function of the gene also in Bacillus subtilis. Determination of the nucleotide sequence of the gene and adjacent DNA indicates that it encodes a polypeptide of 32,903 daltons. A putative promoter for transcription of the gene lies within a hundred nucleotides of the polypeptide start codon. Comparison of the coding sequence to that of the dam gene of Escherichia coli, which encodes a similar methylase, revealed 30% of the amino acid residues in the two enzymes to be identical. This homology presumably reflects a common origin of the two genes prior to the divergence of Gram-positive and Gram-negative bacteria. It is suggested that the restriction function of the gene is primitive, and that the homologous restriction system in E. coli has evolved to play an accessory role in heteroduplex DNA base mismatch repair.

Amino Acid Sequence↗
PubMed1985

Heteroduplex DNA mismatch repair system of Streptococcus pneumoniae: cloning and expression of the hexA gene.

Mutations affecting heteroduplex DNA mismatch repair in Streptococcus pneumoniae were localized in two genes, hexA and hexB, by fractionation of restriction fragments carrying mutant alleles. A fragment containing the hexA4 allele was cloned in the S. pneumoniae cloning system, and the hexA+ allele was introduced into the recombinant plasmid by chromosomal facilitation of plasmid transfer. Subcloning localized the functional hexA gene to a 3.5-kilobase segment of the cloned pneumococcal DNA. The product of this gene was shown in Bacillus subtilis minicells to be a polypeptide with an Mr of 86,000. Two mutant alleles of hexA showed partial expression of the repair system when present in multicopy plasmids. A model for mismatch repair, which depends on the interaction of two protein components to recognize the mismatched base pair and excise a segment of DNA between strand breaks surrounding the mismatch, is proposed.

Alleles↗
PubMed1984

Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis.

The 10-kb chromosomal fragment of Streptococcus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. By the mechanism of chromosomal facilitation, in which the chromosome restores information to an entering plasmid fragment, a BamHI fragment missing the sul-d region of pLS80 established the full-sized plasmid, but with the sul-s allele of the recipient chromosome. A spontaneous deletion beginning approximately 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (approximately 10(-3) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed. Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanilamide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus subtilis↗
PubMed1984

Transfer and expression of recombinant plasmids carrying pneumococcal mal genes in Bacillus subtilis.

The pneumococcal mal recombinant plasmid pLS70, which carries two strong promoters for transcription, could not be transferred and maintained intact in Bacillus subtilis. Although it could be established at low frequency, pLS70 was unstable and was rapidly replaced by deleted forms of the plasmid. A deleted derivative plasmid, pLS69, could be transferred at high frequency and maintained intact. In pLS69 the deletion reduces function of both the malM (amylomaltase) and malX (X-fragment) promoters. This mutant mal plasmid still codes for an intact amylomaltase, and the enzyme is produced in both S. pneumoniae and B. subtilis. The amylomaltase, which is inducible by maltose in S. pneumoniae, is synthesized constitutively in B. subtilis and is localized in the cytosol. Although pLS69 enables S. pneumoniae to grow with maltose, the plasmid did not enhance the ability of B. subtilis to use this sugar, presumably because the latter does not transport free maltose into the cell. Minicells of B. subtilis containing pLS69 synthesized the amylomaltase polypeptide but no X-fragment. In S. pneumoniae carrying pLS69, production of the X-fragment is also reduced more than the amylomaltase, when compared to cells carrying pLS70, which produce equal amounts of the two proteins. Inasmuch as the down promoter mutation leaves unchanged both structural genes, their ribosome-binding sites and -10 and -35 promoter sequences, the unequal effect is attributed to differential reduction in AT composition proximal to the promoters. Vector proteins were revealed in minicells as several bands, all located in the cytosol except for an Mr 35000 polypeptide located in the membrane.

Bacillus subtilis↗
PubMed1984

Generation of deletions in pneumococcal mal genes cloned in Bacillus subtilis.

The pneumococcal recombinant plasmid pLS70, which contains two strong promoters for transcription of the malM and malX genes, is unstable when transferred to Bacillus subtilis, and it gives rise to deleted derivatives. Analysis of proteins produced by the deleted plasmids and restriction mapping of 29 different deletions showed that stabilization in B. subtilis was accompanied by deletions affecting both promoters. Plasmids containing even a single strong promoter were at a selective disadvantage. Nucleotide sequences surrounding the deletions in 10 plasmids were determined. Six different deletions occurred between directly repeated sequences of 3-13 base pairs in length, presumably by a recombination mechanism involving short homologies. Four deletions occurred between sites not contained within repeated sequences. A weak but significant similarity of an 11-base sequence was found surrounding these deletions and the corresponding points of junction in the progenitor plasmids. It is suggested that this sequence may be the recognition site for a topoisomerase-like enzyme that can produce deletions.

Bacillus subtilis↗
PubMed1984

Cloning in Streptococcus pneumoniae of the gene for DpnII DNA methylase.

The gene coding for the pneumococcal DNA adenine methylase that recognizes the sequence 5'-GATC-3' was cloned in a strain of Streptococcus pneumoniae that lacked both restriction endonucleases DpnI and DpnII. The gene was cloned as a 3.7-kilobase fragment of chromosomal DNA from a DpnII-containing strain inserted in both possible orientations in the multicopy plasmid vector pMP5 to give recombinant plasmids pMP8 and pMP10. Recombinant plasmids were selected by their resistance to DpnII cleavage. Cells carrying the recombinant plasmids modified phage in vivo so that it was restricted by DpnI- but not DpnII-containing hosts. They also showed levels of DNA methylase activity five times higher than that in cells of the original DpnII strain. No DpnII activity was observed in the clones; therefore, it was concluded that the insert did not contain an intact DpnII endonuclease gene and that methylation of host DNA did not turn on a latent form of the gene.

Cloning, Molecular↗
PubMed1984

Transfer of recombinant plasmids containing the gene for DpnII DNA methylase into strains of Streptococcus pneumoniae that produce DpnI or DpnII restriction endonucleases.

Plasmid transfer via the transformation pathway of Streptococcus pneumoniae was weakly restricted by the DpnI or DpnII restriction endonuclease, either of which gave a reduction only to 0.4, compared with phage infection, which was restricted to 10(-5). The greater sensitivity of plasmid transfer compared with chromosomal transformation, which was not at all restricted, can be attributed to partially double-stranded intermediates formed from two complementary donor fragments. However, clustering of potential restriction sites in the plasmids increased the probability of escape from restriction. The recombinant plasmid pMP10 , in which the gene for the DpnII DNA methylase was cloned, can be transferred to strains that contain neither restriction enzyme or that contain DpnII as readily as can the vector pMP5 . Introduction of pMP10 raised the level of methylase by five times the level normally present in DpnII strains. Transfer of pMP10 to DpnI -containing strains was infrequent, presumably owing to the suicidal methylation of DNA which rendered it susceptible to the host endonuclease. The few clones in which pMP10 was established had lost DpnI . Loss of the plasmid after curing of the cell eliminated the methylase but did not restore DpnI . Although this loss of DpnI could result from spontaneous mutation, its relatively high frequency, 0.1% suggested that the loss was due to a regulatory shift.

Base Sequence↗
PubMed1984

Ectopic integration of chromosomal genes in Streptococcus pneumoniae.

When a DNA fragment containing a marker gene was ligated to random chromosomal fragments of Streptococcus pneumoniae and used to transform a recipient strain lacking that gene, the gene was integrated at various locations in the chromosome. Such ectopic integration was demonstrated for the malM gene, and its molecular basis was analyzed with defined donor molecules consisting of ligated fragments containing the malM and sul genes of S. pneumoniae. In a recipient strain deleted in the mal region of its chromosome, these constructs gave Mal+ transformants in which the malM and sul genes were now linked, with malM located between duplicate sul segments. Ectopic integration was unstable under nonselective conditions; mal(sul) ectopic insertions were lost at a rate of 0.05% per generation. Several possible mechanisms of ectopic integration were examined. The donor molecule is most likely to be a circular form of ligated homologous and nonhomologous fragments that, after entry into the cell, undergoes circular synapsis with the recipient chromosome at the site of homology, followed by repair and additive integration.

Chromosomes, Bacterial↗
PubMed1983

Isolation and characterization of three new classes of transformation-deficient mutants of Streptococcus pneumoniae that are defective in DNA transport and genetic recombination.

Transformation-deficient mutants of Streptococcus pneumoniae were isolated after nitrosoguanidine mutagenesis. Seventeen mutants developed normal peaks of competence, as tested by their ability to degrade one strand of donor DNA, but they yielded transformants for chromosomal point markers at efficiencies from less than 0.001 to 0.04 that of the wild type. Some of the mutants were defective in DNA uptake and are described as entry defective (Ent-). Others took up DNA in normal quantities, but they failed to give stable transformants and are described as recombination defective (Rec-). In two of the Rec- mutants, normal levels of transformation by plasmid DNA occurred; in the others, it was reduced as much as chromosomal transformation. Conjugative transfers of a chromosomal omega (cat tet) element and of the plasmid pIP501 occurred at normal levels both to and from Rec- mutants. Transfer of chloramphenicol resistance by transformation with omega (cat tet) donor DNA, however, was blocked in Rec- mutants to about the same extent as was transformation for point markers.

Conjugation, Genetic↗
PubMed1982

Identification of base mismatches recognized by the heteroduplex-DNA-repair system of Streptococcus pneumoniae.

The susceptibility to repair of particular base mismatches by the hex system of Streptococcus pneumoniae was examined by comparison of the nucleotide sequence of the wild-type and eight mutant alleles of the malM gene. A detailed restriction map was constructed for pLS70, and the nucleotide sequence was determined for its 3475 bp chromosomal insert, which contains the entire malM gene (encoding amylomaltase), portions of malX and malP (encoding a membrane protein and a phosphorylase, respectively) and a control region. Transition mismatches were highly susceptible to repair; transversion mismatches, much less so. A mismatch caused by a single-nucleotide deletion was reparable, but mismatches with longer deletions were not. The hex system also reduced spontaneous reversion of mutations corresponding to transitions. It is suggested that recognition of donor or nascent DNA strands by the hex system depends on single-strand breaks in the target strand, and that the role of DNA methylation in mismatch repair of Escherichia coli can be accommodated to this model.

Amino Acid Sequence↗
PubMed1982

Effect of strong promoters on the cloning in Escherichia coli of DNA fragments from Streptococcus pneumoniae.

Attempts to clone wild-type DNA containing the malM gene of Streptococcus pneumoniae in plasmid pBR322 of Escherichia coli were unsuccessful. However, it was possible to clone a PstI fragment of DNA containing this gene in a plasmid of S. pneumoniae. Cells carrying the recombinant plasmid produced large amounts of the malM product, amylomaltase, and a fragment of the protein coded by the adjacent malX gene, apparently as a result of transcription in opposite directions from strong promoters located between the two genes in the plasmid insert. Under derepressed conditions these products represented 10% of the total protein. No transcription terminators appeared to be included within the cloned segment. The effect of various mutations in the segment on its ability to be cloned in pBR322 was examined. Of those tested, only a down promoter mutation that affected production of both the amylomaltase and the X-protein rendered the segment clonable in E. coli. Fragments of the S. pneumoniae vector, pMV158, which appear to lack strong promoters, were readily cloned in the pBR322-E. coli system. Although it is possible that large amounts of the X-fragment are toxic for E. coli, a more general explanation would be that excessive transcription of the pBR322 vector portion interferes with maintenance of the recombinant plasmid.

Cloning, Molecular↗
PubMed1982

Nucleotide sequence of DNA controlling expression of genes for maltosaccharide utilization in Streptococcus pneumoniae.

An analysis of previous data indicated that four structural genes concerned with maltosaccharide utilization in Streptococcus pneumoniae are organized in two operons that are transcribed in opposite directions from a central control region. This region contains two strong promoters subject to repression by a regulatory gene product in the absence of maltose. The nucleotide sequence of the 554-bp control region DNA and adjacent portions of the malX and malM structural genes was determined. Unique reading frames and initiation codons allowed identification of the oppositely oriented structural genes. Putative ribosome binding sites and -10 and -35 RNA-polymerase-binding sites, as well as AT-rich regions farther upstream, were observed proximal to both the X and M genes. The similarity of these sequences to sites found in Escherichia coli and Bacillus subtilis indicated the conservation of control signals in bacteria, both Gram-negative and Gram-positive. A pair of 17-bp hyphenated repeat sequences in the control region may represent repressor binding sites. Two down promoter mutations, VII and 69, were shown to be deletions in the control region. The VII mutation, which affected only the MP operon, deleted the promoter adjacent to the M gene. Mutation 69, which reduced both X and M gene functions, deleted the entire segment between the promoters so that they now overlap at their -35 binding sites. As a consequence of this deletion, the AT-rich regions proximal to the promoters were lost. This suggests that the AT-rich regions are important for promoter strength.

Base Sequence↗
PubMed1982

Facilitation of plasmid transfer in Streptococcus pneumoniae by chromosomal homology.

The frequency of plasmid establishment in the transformation of Streptococcus pneumoniae by plasmid DNA was increased more than 10-fold when the plasmid carried DNA homologous to the host chromosome. Perfect homology was not necessary for such facilitation; small additions or deletions were tolerated, but extensive deletions in the homologous segment of either plasmid or chromosome reduced or eliminated facilitation. The facilitated plasmid transfer showed a linear dependence on monomeric plasmid concentration rather than the quadratic dependence found in the absence of homology, which indicated that entering plasmid fragments interacted with the chromosome rather than with each other to establish a plasmid replicon. Restriction enzyme cleavage of the plasmid in the nonhomologous segment destroyed its activity, but cleavage in the homologous segment or even enzymatic removal of part of that segment did not prevent plasmid transfer, and plasmids of the original size were established. In facilitated transfer, chromosomal markers (additions and deletions as well as single-site mutations) entered the plasmid with a frequency ranging from 10 to 90% depending on the marker location. Several possible mechanisms for the establishment of plasmids in the presence of chromosomal homology and for the transfer of chromosomal information are considered. They depend on synapsis of the newly entered single-strand plasmid fragment with the host chromosome and subsequent copying of, donation from, or integration into the homologous chromosomal segment. After plasmid establishment, equilibration of donor and chromosomal markers between the chromosome and the plasmid pool, presumably by homologous recombination events, was observed.

Alleles↗
PubMed1982

Transformation of restriction endonuclease phenotype in Streptococcus pneumoniae.

The genetic basis of the unique restriction endonuclease DpnI, that cleaves only at a methylated sequence, 5'-GmeATC-3', and of the complementary endonuclease DpnII, which cleaves at the same sequence when it is not methylated, was investigated. Different strains of Streptococcus pneumoniae isolated from patients contained either DpnI (two isolates) or DpnII (six isolates). The latter strains also contained DNA methylated at the 5'-GATC-3' sequence. A restrictable bacteriophage, HB-3, was used to characterize the various strains and to select for transformants. One laboratory strain contained neither DpnI nor Dpn II. It was probably derived from a DpnI-containing strain, and its DNA was not methylated at 5'-GATC-3'. Cells of this strain were transformed to the DpnI restriction phenotype by DNA from a DpnI-containing strain and to the DpnII restriction phenotype by DNA from a DpnII-containing strain. Neither cross-transformation, that is, transformation to one phenotype by DNA from a strain of the other phenotype, nor spontaneous conversion was observed. Extracts of transformants to the new restriction phenotype were shown to contain the corresponding endonuclease.

Bacteriophages↗
PubMed1981

Deoxyribonuclease I in mammalian tissues. Specificity of inhibition by actin.

Enzymes of the DNase I class, similar to bovine pancreatic DNase I with respect to molecular weight and ionic and pH requirements, were found in various tissues of the rat. Their analysis was facilitated by a method for detection of nucleases in crude extracts after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and subsequent renaturation of the enzymes. High levels of DNase I were found in digestive tissues, such as the parotid and submaxillary salivary glands and the lining of the small intestine., Appreciable levels were present in the lymph node, kidney, heart, prostate gland, and seminal vesicle. No activity was found in pancreatic extracts. However, under some conditions, tissues rich in proteases gave poor recovery of DNase I. Fourteen other tissues showed little or no DNase I. Inhibition of various DNase I enzymes by rabbit muscle actin was examined both in gels and in solution. Actin inhibited the bovine parotid DNase I as well as the bovine pancreatic enzyme, but actin did not inhibit any of the DNase I enzymes of the rat. This species specificity of actin inhibition makes it unlikely that the very strong association between monomeric actin and bovine DNase I is of general significance for cellular function.

Actins↗
PubMed1981

Nonsense mutations in the amylomaltase gene and other loci of Streptococcus pneumoniae.

Maltose-negative mutations in the amylomaltase gene of Streptococcus pneumoniae were examined for the presence of nonsense mutations. Out of 28 single-site mutants tested, 3 were shown to be suppressible by an amber suppressor previously found by Gasc et al. (1979). In the presence of the suppressor these mutants manifested 10--30% of wild type amylomaltase activity. In addition to the amylomaltase governed by malM, and the maltosaccharide phosphorylase governed by malP (which maps to the side of malM distal to the regulatory gene, malR), a new maltose-inducible protein, governed by another gene, malX, was observed in gel electrophoretic patterns. The malX gene maps on the side of malM proximal to the malR gene. The approximate molecular weights of the amylomaltase, phosphorylase and malX polypeptides are 62,000, 87,000 and 50,000, respectively. There appear to be no polar effects of the nonsense mutations in the malM gene on synthesis of the gene products of either malP or malX. In a search for nonsense mutants at other loci, one was found in the end gene, which governs the major endonuclease, a membrane enzyme. None were detected among 5 mismatch-repair defective hex mutants analyzed.

Electrophoresis, Polyacrylamide Gel↗
PubMed1981

Cloning of chromosomal genes in Streptococcus pneumoniae.

A system for molecular cloning in Streptococcus pneumoniae was developed. The multicopy plasmids pMV158 (5.4 kilobases) and pLS1 (4.3 kilobases), which confer tetracycline resistance, were used as vectors to clone chromosomal genes of S. pneumoniae in host cells of this species. A 3.3-kilobase restriction fragment containing the malM gene, which codes for amylomaltase, was cloned in a deletion mutant lacking chromosomal homology with the fragment. The recombinant plasmid pLS70, could transform over 50% of a recipient population to maltose utilization. Amylomaltase constituted up to 10% of the protein of cells containing pLS70. A derivative with a deletion, pLS69, appeared to gain a selective advantage by producing less enzyme. A 10-kilobase restriction fragment containing the sul-d gene for sulfonamide resistance was cloned in the presence of the homologous chromosomal gene. De novo establishment of a recombinant plasmid was just as frequent as transformation in an endogenous plasmid. Despite the processing of DNA during uptake in the transformation of S. pneumoniae, recombinant plasmids can be introduced. Models for the reconstruction of recombinant DNA in cells of S. pneumoniae and Bacillus subtilis are considered and compared.

Chromosomes, Bacterial↗