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Biomedical subjects

J W Zyskind

Publications and source records attributed to J W Zyskind.

At least 19 recordsLinked to original sources
PubMed1995

Cloning of nod gene regions from mesquite rhizobia and bradyrhizobia and nucleotide sequence of the nodD gene from mesquite rhizobia.

Nitrogen-fixing symbiosis between bacteria and the tree legume mesquite (Prosopis glandulosa) is important for the maintenance of many desert ecosystems. Genes essential for nodulation and for extending the host range to mesquite were isolated from cosmid libraries of Rhizobium (mesquite) sp. strain HW17b and Bradyrhizobium (mesquite) sp. strain HW10h and were shown to be closely linked. All of the cosmid clones of rhizobia that extended the host range of Rhizobium (Parasponia) sp. strain NGR234CS to mesquite also supported nodulation of a Sym- mesquite strain. The cosmid clones of bradyrhizobia that extended the host range of Rhizobium (Parasponia) sp. strain NGR234CS to mesquite were only able to confer nodulation ability in the Sym- mesquite strain if they also contained a nodD-hybridizing region. Subclones containing just the nodD genes of either genus did not extend the host range of Rhizobium (Parasponia) sp. to mesquite, indicating that the nodD gene is insufficient for mesquite nodulation. The nodD gene region is conserved among mesquite-nodulating rhizobia regardless of the soil depth from which they were collected, indicating descent from a common ancestor. In a tree of distance relationships, the NodD amino acid sequence from mesquite rhizobia clusters with homologs from symbionts that can infect both herbaceous and tree legumes, including Rhizobium tropici, Rhizobium leguminosarum bv; phaseoli, Rhizobium loti, and Bradyrhizobium japonicum.

Amino Acid Sequence↗
PubMed1992

DNA replication, the bacterial cell cycle, and cell growth.

The coupling of replication to the cell cycle and cell growth involves events that occur at oriC. Immediately after initiation, there is an eclipse phase during which reinitiation from the newly synthesized origins is prevented. GATC sites in oriC remain in a hemimethylated state longer than other sites because of their association with the outer membrane, which prevents DnaA from binding and activating additional rounds of initiation. After the origins are methylated and released from the outer membrane, the concentration of newly synthesized DnaA and the activation of oriC by transcription from the nearby mioC and gid promoters determine when the next rounds of replication initiate. If growth rate is reduced, the synthesis of (p)ppGpp will increase, and this will lead to a decrease in dnaA, mioC, and gid transcription. On the other hand, if growth rate is increased by access to a tasty meal, synthesis of (p)ppGpp will decrease, expression of dnaA, mioC, and gid genes will increase, and a shortening of the interinitiation time will result. The participation of all these control features ensures rapid and precise coordination of DNA replication with cell growth.

Bacteria↗
PubMed1992

RecA protein of Escherichia coli and chromosome partitioning.

Escherichia coli cells deficient in RecA protein frequently contain an abnormal number of chromosomes after completion of ongoing rounds of DNA replication. This suggests that RecA protein may be required for correct timing of initiation of DNA replication; however, we show here that initiation of DNA replication is properly timed in recA mutants. We also find that more than 10% of recA mutant cells contain no DNA. These anucleate cells appear to arise from partitioning of all the DNA into one daughter cell and no DNA into the other daughter cell. Based on these and previously published results, we propose that RecA protein is required for equal partitioning of chromosomes into the two daughter cells.

Cell Cycle↗
PubMed1992

Escherichia coli cells lacking methylation-blocking factor (leucine-responsive regulatory protein) have precise timing of initiation of DNA replication in the cell cycle.

A protein that is required for specific methylation inhibition of two GATC sites in the papBA pilin promoter region, known as methylation-blocking factor (Mbf) and recently shown to be identical to the leucine-responsive regulatory protein (Lrp), is not responsible for the delayed methylation at oriC implicated in an eclipse period following initiation of DNA replication. Cells containing a transposon mutation within the mbf (lrp) gene initiate DNA replication at the correct time during the cell cycle, whereas cells with increased amounts of the Dam methyltransferase initiate DNA replication randomly throughout the cell cycle.

Bacterial Proteins↗
PubMed1990

Coupling of DNA replication to growth rate in Escherichia coli: a possible role for guanosine tetraphosphate.

Two promoters for the Escherichia coli operon that contains the four genes dnaA, dnaN, recF, and gyrB were found to be growth rate regulated and under stringent control. Transcript abundance relative to total RNA increased with the growth rate. Changes in transcription from the dnaAp1 and dnaAp2 promoters that were induced by amino acid starvation and chloramphenicol and were relA dependent were correlated with the stringent response. The abundance of these transcripts per total RNA also decreased in spoT mutants as the severity of the mutation increased (guanosine 5'-diphosphate 3'-diphosphate [ppGpp] basal levels increased). Because expression of these promoters appears to be inhibited by ppGpp, it is proposed that one mechanism for coupling DNA replication to the growth rate of bacteria is through ppGpp synthesis at the ribosome.

DNA Replication↗
PubMed1989

A model for the initiation of replication in Escherichia coli.

The role of the protein DnaA as the principal control of replication initiation is investigated by a mathematical model. Data showing that DnaA is growth rate regulated suggest that its concentration alone is not the only factor determining the timing of initiation. A mathematical model with stochastic and deterministic components is constructed from known experimental evidence and subdivides the total pool of DnaA protein into four forms. The active form, DnaA.ATP, can be bound to the origin of replication, oriC, where it is assumed that a critical level of these bound molecules is needed to initiate replication. The active form can also exist in a reserve pool bound to the chromosome or a free pool in the cytoplasm. Finally, a large inactive pool of DnaA protein completes the state variables and provides an explanation for how the DnaA.ATP form could be the principal controlling element in the timing of initiation. The fact that DnaA protein is an autorepressor is used to derive its synthesis rate. The model studies a single exponentially growing cell through a series of cell divisions. Computer simulations are performed, and the results compare favorably to data for different cell cycle times. The model shows synchrony of initiation events in agreement with experimental results.

Adenosine Triphosphate↗
PubMed1989

N,N'-diacetylchitobiase of Vibrio harveyi. Primary structure, processing, and evolutionary relationships.

The nucleotide sequence of the gene, chb, encoding the outer membrane protein, N,N'-diacetylchitobiase (chitobiase), of the marine bacterium, Vibrio harveyi, has been determined. The amino acid sequence of prechitobiase was derived from the nucleotide sequence. Prechitobiase has a molecular mass of 97,771 Da and consists of 883 amino acid residues. A characteristic signal peptide is present at the amino terminus whose removal is inhibited by the antibiotic, globomycin, suggesting that mature chitobiase is a lipoprotein with a maturation pathway similar to that of the Escherichia coli major outer membrane lipoprotein. A perfect homology to six amino acids at the processing and modification region of the outer membrane lipoprotein of E. coli was found with amino acids 15-19 of the deduced prechitobiase protein sequence. Chitobiase shares similarities and possibly common ancestry with the alpha-chain of the human beta-hexosaminidase. A comparison of the amino acid sequences of chitobiase and the alpha-chain of beta-hexosaminidase gave a highly significant alignment score of 19.1 standard deviation units above a mean randomized alignment score. Primer extension analysis of the promoter region revealed three transcription initiation sites used by E. coli cells harboring the chb gene, two of which were also evident in V. harveyi cells.

Acetylglucosaminidase↗
PubMed1989

Expression of Escherichia coli dnaA and mioC genes as a function of growth rate.

The synthesis of specific cellular components related to the initiation process of DNA replication was correlated with changes in growth rate. The concentrations of DnaA protein and mioC mRNA were determined for cells grown at six different growth rates; both increased relative to either total protein or total RNA, respectively, as the growth rate increased. Expression from the chromosomal mioC promoter, which contains a DnaA protein-binding site, was not repressed when the DnaA protein concentration was increased and was not derepressed in a dnaA46 mutant at 42 degrees C. The mioC transcript had a characteristic mRNA-type half-life of 1.51 min.

Bacterial Proteins↗
PubMed1987

RNA polymerase pauses in vitro within the Escherichia coli origin of replication at the same sites where termination occurs in vivo.

An in vitro transcription system able to distinguish initiation at the 16-kDa promoter from elongation events was used to identify factors that might participate in transcription termination within oriC. Pausing in the oriC region occurs at the same sites where termination occurs in vivo. Ten of these sites overlap RNA:DNA junctions in oriC. The pausing that occurs in vitro was not converted to efficient termination by guanosine 5'-diphosphate 3'-diphosphate, NusA, and Rho alone or in combination, or by DnaA suggesting that in vivo other or additional factors contribute to termination at oriC. Transcription from the 16-kDa promoter was 90% inhibited by the nucleotides guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate in agreement with previous observations that this promoter is stringently regulated.

Bacterial Proteins↗
PubMed1987

Translocation of Vibrio harveyi N,N'-diacetylchitobiase to the outer membrane of Escherichia coli.

The gene encoding N,N'-diacetylchitobiase (chitobiase) of the chitinolytic marine bacterium Vibrio harveyi has been isolated. While expression of the chitobiase gene (chb) was inducible by N,N'-diacetylchitobiose in V. harveyi, it was expressed constitutively when cloned in Escherichia coli, suggesting that controlling elements are not closely linked to chb. Chitobiase was found in the membrane fraction of E. coli cells containing plasmids with the cloned V. harveyi chb gene. When membranes of such cells were separated on Osborn gradients, chitobiase activity was found mainly in the outer membrane band. Translocation of the enzyme to the outer membrane was accompanied by cleavage of a signal peptide. A fusion protein, in which 22 amino acids from the amino terminus of prechitobiase were replaced with 21 amino acids from the pUC19 lacZ amino terminus, was not processed, and 99% of the activity was located in the cytoplasmic fraction. A homology to six amino acids surrounding the lipoprotein processing and modification site was found near the amino terminus of prechitobiase.

Acetylglucosaminidase↗
PubMed1986

RNA terminating within the E. coli origin of replication: stringent regulation and control by DnaA protein.

RNA entering the E. coli replication origin, oriC, in the counterclockwise direction terminates at several sites throughout the origin sequence. The significant finding was that nine clusters of these termination sites are found at the nine clusters of RNA to DNA transitions in oriC. The majority of these transcripts terminates with cytosine. Termination sites are associated with 9 of the 11 GATC sites and all DnaA protein-binding sites. Chloramphenicol-treated cells contain an increased amount of this RNA species, while cells starved for isoleucine have greatly reduced levels, indicating that synthesis of these transcripts is stringently regulated. Both decreased and increased intracellular levels of DnaA protein decrease the fraction of transcription that enters oriC.

Bacterial Proteins↗
PubMed1986

Transcription termination within the Escherichia coli origin of DNA replication, oriC.

Initiation of DNA replication from the Escherichia coli origin, oriC, is dependent on an RNA polymerase-mediated transcription event. The function of this RNA synthetic event in initiation, however, remains obscure. Since control of the synthesis of this RNA could serve a key role in the overall initiation process, transcription regulatory sites within and near oriC were identified using the galK fusion vector system. Our results confirm the existence of a transcription termination signal within oriC, first identified by Hansen et al. (1981), for the 16 kd transcript that is transcribed counterclockwise towards oriC. Termination is shown to be 92% efficient. A similar approach led to the detection of transcription termination within the chromosomal replication origin of Klebsiella pneumoniae. Approximately 50% of the E. coli 16 kd transcripts appear to terminate before reaching oriC between the XhoI (+416 bp) and the HindIII (+243 bp) sites. The predominant 3' ends of RNA that enter oriC, as determined by SI nuclease mapping, were located at positions +20 +/- 2, +23 +/- 2, +37, +39, +52, +66, +92, and +107. These termination sites, which map cl to RNA . DNA junctions identified by Kohara et al. (1985), appear as triplets and quadruplets. The E. coli oriC Pori-L promoter described in in vitro transcription studies by Lother and Messer (1981) was not detected in this study in either wildtype cells or isogenic dnaA mutants at the nonpermissive temperature. A new promoter activity, Pori-R1, was identified within the E. coli origin in the clockwise direction.

DNA Replication↗
PubMed1985

Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli.

In vivo and in vitro evidence is presented implicating a function of GATC methylation in the Escherichia coli replication origin, oriC, during initiation of DNA synthesis. Transformation frequencies of oriC plasmids into E. coli dam mutants, deficient in the GATC-specific DNA methylase, are greatly reduced compared with parental dam+ cells, particularly for plasmids that must use oriC for initiation. Mutations that suppress the mismatch repair deficiency of dam mutants do not increase these low transformation frequencies, implicating a new function for the Dam methylase. oriC DNA isolated from dam- cells functions 2- to 4-fold less well in the oriC-specific in vitro initiation system when compared with oriC DNA from dam+ cells. This decreased template activity is restored 2- to 3-fold if the DNA from dam- cells is first methylated with purified Dam methylase. Bacterial origin plasmids or M13-oriC chimeric phage DNA, isolated from either base substitution or insertion dam mutants of E. coli, exhibit some sensitivity to digestion by DpnI, a restriction endonuclease specific for methylated GATC sites, showing that these dam mutants retain some Dam methylation activity. Sites of preferred cleavage are found within the oriC region, as well as in the ColE1-type origin.

DNA Replication↗
PubMed1983

Chromosomal replication origin from the marine bacterium Vibrio harveyi functions in Escherichia coli: oriC consensus sequence.

The chromosomal replication origin (oriC) of Vibrio harveyi has been isolated on a plasmid and shown to function as an origin in Escherichia coli. The nucleotide sequence of the V. harveyi oriC was determined. From a comparison of this sequence with oriC sequences of five enteric bacteria, we derived a consensus sequence of bacterial origins that function in E. coli. This consensus sequence identifies 122 positions within oriC where nucleotide substitutions can occur without loss of origin function. These positions are clustered rather than scattered. Four interrelated nine-base-pair repeats and eight of the dam methylation G-A-T-C sites are conserved in the consensus sequence. Very few relative insertion-deletion changes occur, and these are localized to one region of oriC. The genes for three polypeptides linked to the V. harveyi oriC were identified by using in vitro protein synthesis directed by deletion derivative plasmid templates. One of these genes, coding for a 58,000 Mr polypeptide and located 3.0 kilobase pairs from the V. harveyi oriC region, is lethal to E. coli when many copies (approximately 40 per cell) are present (high copy lethal or HCL gene). In addition, nucleotide sequence analysis showed that a different gene, the gid gene to the left of oriC, is highly conserved between E. coli and V. harveyi, whereas the coding region to the right of oriC is much less conserved.

Base Sequence↗
PubMed1982

The chromosomal origin of replication (oriC) of Erwinia carotovora.

The chromosomal DNA replication origin (oriC) of the plant pathogen Erwinia carotovora has been isolated and sequenced. The minimal E. carotovora oriC regional functional in Escherichia coli is a 374 base pair region located on a 7.9 kilobase pair SalI fragment which also contains a functional asnA gene. Differences between the nucleotide sequence of the minimal origin regions of E. carotovora and those of E. coli and Salmonella typhimurium are clustered nucleotide substitutions, with regions of complete homology, up to 19 base pairs long, between the three origins. Nine GATC sites are found in the minimal origin, and all are conserved. In contrast, the region toward asnA from the minimal origin shows little clustering and the differences occur mainly every third nucleotide, suggesting that this region is a protein coding region.

Base Sequence↗
PubMed1982

Primary structure of the chromosomal origins (oriC) of Enterobacter aerogenes and Klebsiella pneumoniae: comparisons and evolutionary relationships.

The nucleotide sequences of the Enterobacter aerogenes and Klebsiella pneumoniae DNA replication origins (oriC) were determined and compared with those of Escherichia coli and Salmonella typhimurium. Four interrelated, 9-base-pair repeats were identified from the conserved regions within the minimal origin. Evolutionary rates calculated from the minimal origin sequences yielded a quantitative phylogenic tree which agreed with the taxonomic classification of these genera.

Base Sequence↗
PubMed1982

Chromosomal replication origins (oriC) of Enterobacter aerogenes and Klebsiella pneumoniae are functional in Escherichia coli.

The chromosomal DNA replication origins (oriC) from two members of the family Enterobacteriaceae, Enterobacter aerogenes and Klebsiella pneumoniae, have been isolated as functional replication origins in Escherichia coli. The origins in the SalI restriction fragments of 17.5 and 10.2 kilobase pairs, cloned from E. aerogenes and K. pneumoniae, respectively, were found to be between the asnA and uncB genes, as are the origins of the E. coli and Salmonella typhimurium chromosomes. Plasmids containing oriC from E aerogenes, K. pneumoniae, and S. typhimurium replicate in the E. coli cell-free enzyme system (Fuller, et al., Proc. Natl. Acad. Sci. U.S.A. 78:7370--7374, 1981), and this replication is dependent on dnaA protein activity. These SalI fragments from E. aerogenes and K. pneumoniae carry a region which is lethal to E. coli when many copies are present. We show that this region is also carried on the E. coli 9.0-kilobase-pair EcoRI restriction fragment containing oriC. The F0 genes of the atp or unc operon, when linked to the unc operon promoter, are apparently responsible for the lethality.

Bacterial Proteins↗