Biomedical subjects

A Gruss

Publications and source records attributed to A Gruss.

At least 55 records · Page 3Linked to original sources

PubMed1995

A 7-base-pair sequence protects DNA from exonucleolytic degradation in Lactococcus lactis.

Linear DNA molecules are subject to degradation by various exonucleases in vivo unless their ends are protected. It has been demonstrated that a specific 8-bp sequence, 5'-GCTGGTGG-3', named Chi, can protect linear double-stranded DNA from the major Escherichia coli exonuclease RecBCD. Chi protects linear replication products of rolling-circle plasmids from RecBCD degradation in vivo, in agreement with observations in vitro. A unique 7-bp sequence, 5'-GCGCGTG-3', is shown to protect similar replication products from degradation in Lactococcus lactis strains but not in more distantly related Gram-positive bacteria. The properties of this sequence in L. lactis correspond to those of a Chi site. Linear plasmid replication products have been detected in numerous prokaryotes, suggesting the widespread existence of short species-specific sequences that preserve linear DNA from extensive degradation by host cell exonucleases.

Base Composition↗

PubMed1995

The recA gene of Lactococcus lactis: characterization and involvement in oxidative and thermal stress.

The role of recA in Lactococcus lactis, a microaerophilic fermenting organism, was examined by constructing a recA-disrupted strain. This single alteration had a surprisingly pleiotropic effect. In addition to its roles in homologous recombination and DNA repair, recA is also involved in responses to oxygen and heat stresses. We found that oxygen stress induced by aeration causes reductions in growth and stationary-phase survival of the recA strain. Toxicity is a consequence of hydroxyl radical production via the Fenton Reaction and is alleviated by catalase or Ferrozine addition. These results suggest that oxygen radicals are not efficiently eliminated and accumulate in lactococcal cultures, and that RecA is needed to deal with the damage they incur. Unexpectedly, thermal stress arrested growth of the recA strain. Immunological data indicate that the recA mutant is deficient in heat-shock proteins DnaK, GroEL, and GrpE. Poor growth at elevated temperature is therefore due to a diminished heat-shock response in the recA strain. In contrast, levels of a novel heat-shock protein, HfIB, are elevated. In Escherichia coli, HfIB downregulates the heat-shock response by promoting degradation of the transcription factor sigma 32. We propose that recA regulates the heat-shock response via HfIB. This work provides the first evidence showing that two major pathways of stress response, induced by heat shock and DNA damage, are interactive.

ATP-Dependent Proteases↗

PubMed1995

recA gene involvement in oxidative and thermal stress in Lactococcus lactis.

The recA gene is best known for its effects on homologous recombination and DNA repair via SOS induction. There is gathering evidence that recA also affects expression of genes associated with different types of stress. We studied recA properties in Lactococcus lactis by generating a recA-disrupted mutant of MG1363 and comparing it with the wild type strain. recA appears to have an important role in cell survival upon oxygen or thermal stress, in addition to its conserved role in DNA repair. Oxygen toxicity appears to be due to the production of hydroxyl radicals via the Fenton reaction; recA would be involved in the repair of DNA damage generated by these radicals. Surprisingly, the recA strain stops growing at elevated temperature (37 degrees C). Immunological tests indicate that amounts of three heat shock proteins are reduced in the recA strain compared to the wild type strain. In contrast, the amount of heat shock regulator HflB is markedly increased, even at low temperature. HflB is known to degrade heat shock transcription factor sigma 32 in Escherichia coli. We propose that heat shock response is reduced in the recA mutant due to overproduction of HflB.

ATP-Dependent Proteases↗

PubMed1994

Vaginal clear cell carcinoma in a young patient with ectopic termination of the left ureter in the vagina.

The association of clear cell adenocarcinoma of the vagina and vaginal adenosis with prenatal exposure to diethylstilbestrol (DES) is well-documented in the United States. In Europe, however, DES was never used in the therapy of threatened abortion and, therefore, clear cell adenocarcinoma and vaginal adenosis remained rare diseases. We report on the clinical and pathological features of a case of clear cell adenocarcinoma of the upper vagina in a 17-year-old German girl, who had a history of hypoplasia of the left kidney with an ectopic termination of the ureter in the upper vagina, removed surgically 2 years before. No previous report of a similar coincidence of vaginal clear cell carcinoma and a congenital disorder of the genitourinary tract exists. Congenital anomaly of the ureter interfering with the development and the differentiation of the distal Müllerian tract and its epithelium might have provided a similar histological basis for carcinogenesis in our patient to that in those provided exposed to DES.

Adenocarcinoma, Clear Cell↗

PubMed1994

Direct screening of recombinants in gram-positive bacteria using the secreted staphylococcal nuclease as a reporter.

A system for direct screening of recombinant clones in Lactococcus lactis, based on secretion of the staphylococcal nuclease (SNase) in the organism, was developed. The nuc gene (encoding SNase) was cloned on both rolling-circle and theta-replicating plasmids. L. lactis strains containing these nuc+ plasmids secrete SNase and are readily detectable by a simple plate test. A multicloning site (MCS) was introduced just after the cleavage site between leader peptide and the mature SNase, without affecting nuclease activity. Cloning foreign DNA fragments into any site of the MCS interrupts nuc and thus results in nuc mutant clones which are easily distinguished fron nuc+ clones on plates. The utility of this system for L. lactis was demonstrated by cloning an antibiotic resistance marker and Escherichia coli chromosomal DNA fragments into the MCS of the nucMCS cassette. Both cloning vectors containing the nucMCS cassette were also introduced into Streptococcus salivarius subsp. thermophilus, in which direct screening of nuc mutant recombinant clones was also achieved. The potential uses of nuc as a secretion reporter system are discussed.

Base Sequence↗

PubMed1993

High-efficiency gene inactivation and replacement system for gram-positive bacteria.

A system for high-efficiency single- and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG+host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37 degrees C. A nested set of L. lactis chromosomal fragments cloned onto pG+host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37 degrees C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28 degrees C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40% of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.

DNA Replication↗

PubMed1993

Mutation in the plasmid pUB110 Rep protein affects termination of rolling circle replication.

We isolated a mutant of plasmid pUB110 that has the following properties in Bacillus subtilis: (i) it is toxic for recA and add cells, particularly at elevated temperature; (ii) it has a copy number threefold higher than that of the parental plasmid, and the extra copies are present as multimers; and (iii) it can efficiently complement replication of a cmp- satellite plasmid, despite being cmp+. All these properties are due to a single change in the plasmid replication protein, i.e., Gly at position 148 to Glu. These properties of the mutant Rep protein reflect a diminished ability to terminate rolling circle replication. We propose that the Rep protein may have a diminished affinity for the plasmid origin; alternatively, it may be impaired for recognition of the plasmid conformations which distinguish initiation and termination.

Amino Acid Sequence↗

PubMed1993

Efficient plasmid mobilization by pIP501 in Lactococcus lactis subsp. lactis.

pIP501 is a streptococcal conjugative plasmid which can be transmitted among numerous gram-positive strains. To identify a minimal mobilization (mob) locus of pIP501, DNA fragments of pIP501 were cloned into nonconjugative target plasmids and tested for mobilization by pIP501. We show that nonmobilizable plasmids containing a specific fragment of pIP501 are transmitted at high frequencies between Lactococcus lactis subsp. lactis strains if transfer (tra) functions are provided in trans by a pIP501 derivative. Independent transfer of the mobilized plasmid was observed in up to 44% of transconjugants. A 2.2-kb segment containing mob was sequenced. This DNA segment is characterized by three palindromes (palI, palII, and palIII) and a 202-amino-acid open reading frame (ORFX) of unknown function. The smallest DNA fragment conferring high frequency mobilization was localized to a 1.0-kb region (extending from pIP501 coordinates 3.60 to 4.60 on the 30.2-kb map) which contains palI (delta G = -27 kcal/mol [ca. -110,000 J/mol]). A 26-bp sequence identical to palI is present on pIP501, upstream of the plasmid copy control region. Further homologies with the palI sequence are also found with the related Enterococcus faecalis conjugative plasmid pAM beta 1. The region containing mob maps outside the previously described segment mediating pIP501 conjugation. Our results with recA strains indicate that the mob site is a hot spot for cointegrate formation.

Amino Acid Sequence↗

PubMed1992

Chi sequence protects against RecBCD degradation of DNA in vivo.

RecBCD is a multifunctional enzyme involved in DNA degradation and homologous recombination. It also produces an endonucleolytic cleavage near properly oriented chi sites (5'-GCTGGTGG-3'). Plasmids are not known to be affected by either RecBCD enzyme or the presence of a chi site. We report here that plasmids that replicate by a rolling circle mechanism accumulate large amounts of high molecular weight linear multimers (HMW), either if they contain a chi site or if RecBCD is absent. An in vivo inducible system for rolling circle replication was constructed to study RecBCD and its interactions with chi. Results show that (i) HMW accumulation is chi orientation dependent, and (ii) a succession of chi sites prevents degradation of HMW by RecBCD enzyme. These results demonstrate chi activity in plasmids. The rolling circle mechanism produces a sigma structure during plasmid replication; we propose that the double-stranded DNA tail of this sigma form allows RecBCD entry; the tail is degraded unless it is protected by a chi site. By analogy, a principal role of chi in the survival of lambda red-gam- mutants in wild-type strains may be to protect rolling circle concatemers (in late replication) from degradation by RecBCD.

Base Sequence↗

PubMed1992

Use of degenerate primers for polymerase chain reaction cloning and sequencing of the Lactococcus lactis subsp. lactis recA gene.

Two particularly well-conserved stretches in the RecA protein sequences were chosen as templates to synthesize degenerate oligonucleotides, which were used in polymerase chain reaction to amplify an internal recA DNA fragment of Lactococcus lactis subsp. lactis ML3. Using this fragment, we recovered and sequenced the entire lactococcal recA gene. The end of an open reading frame present upstream of the recA gene shows strong homology with formamidopyrimidine-DNA-glycosylase, a protein involved in DNA repair.

Amino Acid Sequence↗

PubMed1992

High-molecular-weight linear multimer formation by single-stranded DNA plasmids in Escherichia coli.

We inserted foreign DNA segments into plasmids which replicate by a rolling-circle mechanism in Escherichia coli and observed the appearance of high-molecular-weight plasmid multimers (HMW). This phenomenon, which occurs more frequently with GC-rich segments, depends on the mode of replication of the plasmid and on host homologous recombination functions. We found that (i) HMW are formed upon insertion of a foreign DNA segment into a single-stranded DNA plasmid, whereas the same DNA insert has no such effect on a theta replicon, and (ii) HMW are not present in a recA mutant strain but are found in a lexA (Ind-) mutant. Enzymatic studies allowed us to define the HMW structure as linear double-stranded tandem head-to-tail plasmid repeats. Use of heteroplasmid strains showed that HMW production by one plasmid does not affect another resident plasmid, indicating that no host functions are phenotypically inactivated. This distinguishes our system from the HMW observed with various replicons in the absence of RecBCD enzyme activity. We propose that the role of the foreign insert is to protect the DNA from RecBCD exonuclease attack.

DNA Mutational Analysis↗

PubMed1992

A general method for cloning recA genes of gram-positive bacteria by polymerase chain reaction.

An internal fragment of the recA gene from eight gram-positive organisms has been amplified by using degenerate primers in a polymerase chain reaction. The internal 348- or 360-bp recA DNA segments from Bacillus subtilis, Clostridium acetobutylicum, Lactobacillus bulgaricus, Lactobacillus helveticus, Leuconostoc mesanteroides, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus salivarus subsp. thermophilus were amplified, cloned, and sequenced. The G + C contents of the DNA from these species range from 28 to 52%. The sequences of the bacterial recA genes show strong relatedness. This method is particularly useful for the recovery of the recA genes of gram-positive bacteria and avoids the difficulties of using a genetic complementation test for cloning.

Amino Acid Sequence↗

PubMed1992

New thermosensitive plasmid for gram-positive bacteria.

We isolated a replication-thermosensitive mutant of the broad-host-range replicon pWV01. The mutant pVE6002 is fully thermosensitive above 35 degrees C in both gram-negative and gram-positive bacteria. Four clustered mutations were identified in the gene encoding the replication protein of pVE6002. The thermosensitive derivative of the related plasmid pE194 carries a mutation in the analogous region but not in the same position. Derivatives of the thermosensitive plasmid convenient for cloning purposes have been constructed. The low shut-off temperature of pVE6002 makes it a useful suicide vector for bacteria which are limited in their own temperature growth range. Using pVE6002 as the delivery vector for a transposon Tn10 derivative in Bacillus subtilis, we observed transposition frequencies of about 1%.

Amino Acid Sequence↗

PubMed1991

GC-rich DNA sequences block homologous recombination in vitro.

The capacity of the RecA protein of Escherichia coli to promote an essential step in homologous recombination, strand transfer, was tested on DNA substrates varying in percentage GC. GC content was determined by a novel method using the polymerase chain reaction. Strand transfer activity is greatly reduced as a function of increasing GC content of the DNA. Some reduction is observed with substrates having a GC percentage similar to that of E. coli. The transfer reaction between sequences adjacent, but not distal, to GC-rich sequences is similarly decreased, suggesting that the structure of RecA-DNA complexes may differ with the GC content of DNA. Our results implicate an important role of DNA sequence in homologous recombination and suggest that many sequences are excluded due to their GC content.

Base Composition↗

PubMed1990

Plus-origin mapping of single-stranded DNA plasmid pE194 and nick site homologies with other plasmids.

Staphylococcus aureus plasmid pE194 manifests a natural thermosensitivity for replication and can be established in several species, both gram positive and gram negative, thus making it attractive for use as a delivery vector. Like most characterized plasmids of gram-positive bacteria, pE194 generates single-stranded DNA. The direction of pE194 replication is clockwise, as determined by the strandedness of free single-stranded DNA. Significant homology exists between a 50-base-pair sequence in the origin of pE194 and sequences present in plasmids pMV158 (Streptococcus agalactiae), pADB201 (Mycoplasma mycoides), and pSH71 (Lactococcus lactis). We used an initiation-termination reaction, in which pE194 initiates replication at its own origin and is induced to terminate at the related pMV158 sequence, to demonstrate that pE194 replicates by a rolling-circle mechanism; the initiation nick site was localized to an 8-base-pair sequence.

Base Sequence↗

PubMed1990

Diagnostic value of transrectal ultrasound in tumor staging and in the detection of incidental prostatic cancer.

To evaluate the accuracy of transrectal ultrasonography in staging and detection of prostatic cancer, a prospective study was initiated. In 466 patients transrectal ultrasonography was performed before transurethral resection, suprapubic adenomectomy, or radical prostatectomy. The findings were compared with the histopathologic results. In 160 of the 466 patients a carcinoma of the prostate was proven by histology. In 21 patients the cancer was an incidental finding after transurethral resection. In 133 of the 160 patients (83.1%) the tumor was detected by ultrasound prior to surgery. The analysis of the results revealed, however, great differences in the accuracy in the different tumor stages. In those patients where the tumor was confined to the prostate, the carcinoma was identified only in 37.5% of the cases. Especially prostatic cancer stage pT1a-b (incidental) was detected only in 3 out of 21 patients. Thus, it can be concluded that transrectal ultrasonography is not more sensitive than the rectal palpation for detection of prostatic cancer stage T1a-b.

Carcinoma↗

PubMed1989

Replication origins of single-stranded-DNA plasmid pUB110.

The two replication origins of plasmid pUB110 have been characterized. The site of initiation of DNA replication at the plus origin was mapped to within an 8-base-pair sequence. DNA synthesis initiated at the origin was made to terminate precociously in an inserted sequence of 18 base pairs that is homologous to a sequence in the origin. This suggests that pUB110 replicates as a rolling circle. The minus origin of plasmid pUB110 has been characterized, and the minimal sequence required for function has been determined. As with other minus origins, activity is orientation specific with respect to the direction of replication. Its activity is sensitive to rifampin in vivo, suggesting that RNA polymerase catalyzes single-strand to double-strand conversion. Unlike all other plasmids of gram-positive bacteria thus far described, the pUB110 minus origin is functional in more than one host.

Base Sequence↗

PubMed1989

The family of highly interrelated single-stranded deoxyribonucleic acid plasmids.

Many plasmids from gram-positive bacteria replicate via a single-stranded deoxyribonucleic acid (ssDNA) intermediate, most probably by a rolling-circle mechanism (these plasmids are referred to in this paper as ssDNA plasmids). Their plus and minus origins are physically separated, and replicative initiations are not simultaneous; it is this feature that allows visualization of ssDNA replication intermediates. The insertion of foreign DNA into an ssDNA plasmid may provoke a high frequency of deletions, changes of replicative products to high-molecular-weight forms, segregational loss, and decreased plasmid copy numbers. When an ssDNA plasmid is inserted into the chromosome, both deletions and amplifications may be induced. Both the mode of replication and the copy control mechanism affect the fate of inserted foreign material, usually selecting for its loss. Thus, after having tasted various morsels of DNA, the resulting plasmid stays trim. The features of the ssDNA plasmids seem to be beneficial for their viability and propagation, but not for their use as cloning vectors. However, plasmids replicating via ssDNA intermediates are being exploited to yield insights into the mechanisms of recombination and amplification.

Base Sequence↗