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Biomedical subjects

L Shapiro

Publications and source records attributed to L Shapiro.

At least 235 records · Page 13Linked to original sources
PubMed1985

Organization and nucleotide sequence analysis of an rRNA and tRNA gene cluster from Caulobacter crescentus.

rRNA genes of Caulobacter crescentus CB13 were isolated and shown to be present in two gene clusters in the genome. The organization of each rRNA gene cluster was found to be 5'-16S-tRNA spacer-23S-5S-3'. The DNA sequence of 40% of the 16S rRNA gene, the entire 16S/23S intergenic spacer region, and portions of the 23S rRNA gene were determined. Analysis of the nucleotide sequence in the 16S-23S intergenic spacer region revealed the presence of tRNAIle and tRNAAla genes. Large invert repeat sequences were found surrounding the 16S rRNA gene. These inverted repeat sequences are analogous to the RNase III-processing sites in the E. coli rRNA precursor. Small invert repeat sequences were also found flanking the individual tRNA genes. RNA polymerase-binding studies with restriction fragments of the rRNA gene cluster revealed three regions which bound enzyme, and these regions were shown to contain transcription initiation sites. One of these sites was located within the 16S gene near its 3' end, and the other two were found at the 5' end of the 23S gene.

Base Sequence↗
PubMed1985

Phosphorylation of the beta' Subunit of RNA Polymerase and Other Host Proteins upon phiCd1 Infection of Caulobacter crescentus.

A protein kinase activity is induced early after infection of Caulobacter crescentus by the DNA phage phiCd1. After phage infection at least 40 proteins are phosphorylated; these include DNA-binding proteins, a membrane-associated protein, and several ribosomal proteins. One of the phosphorylated DNA-binding proteins was identified as the beta' subunit of the host RNA polymerase.

Journal Article↗
PubMed1984

Differential expression and positioning of chemotaxis methylation proteins in Caulobacter.

Proteins involved in chemotaxis methylation reactions have been identified in Caulobacter crescentus and their activities, times of synthesis and cellular positions have been determined. The methyl-accepting chemotaxis proteins, the methyl-transferase and the methylesterase were all shown to be active in the flagella-bearing swarmer cell, but all three activities were lost after the swarmer cells shed their flagellum and differentiated into a stalked cell. The membrane methyl-accepting chemotaxis proteins were shown to be synthesized before cell division, coincident with the synthesis of the components of the flagellum, and to be specifically localized in the membrane of the incipient swarmer cell portion of the predivisional cell. The cytoplasmic methylesterase was also found to be differentially synthesized coincident with the period of flagellar biogenesis. Furthermore, methyltransferase activity, present in the predivisional cell, was detected only in the swarmer cell upon cell division. These results demonstrate that the chemotaxis methylation machinery is positionally biased toward one portion of the predivisional cell, and that the time of expression of a set of fla and che genes is correlated with the positioning of their gene products within the cell.

Autoradiography↗
PubMed1984

Generation of a Tn5 promoter probe and its use in the study of gene expression in Caulobacter crescentus.

A promoter probe, Tn5-VB32, was constructed and placed in a P group R plasmid containing bacteriophage Mu sequences, allowing transfer of the transposon to bacteria such as Caulobacter, Rhizobium, and Agrobacterium without retention of the plasmid. The probe carries an altered Tn5 transposon that allows detection of chromosomal promoter regions by virtue of acquired kanamycin resistance. A fragment of DNA containing the neomycin phosphotransferase II (NPT II) gene from Tn5, lacking its promoter region but retaining its translation initiation signal, was inserted into a Tn5 derivative that lacked the entire NPT II gene and a large portion of the IS50L sequence while retaining its ability to transpose. This Tn5 derivative also contained the intact tetracycline resistance-encoding region of the transposon Tn10. Transposition of the Tn5-VB32 promoter probe into the Caulobacter crescentus chromosome generated auxotrophic and motility mutants and Southern blot analysis of DNA from these mutants showed Tn5-VB32 sequences in random-sized chromosomal restriction fragments. Transcriptional regulation by exogenous cysteine of NPT II gene expression was demonstrated in a cysteine auxotroph generated by Tn5-VB32 insertional inactivation. NPT II synthesis, measured by agar plate assays of kanamycin resistance and by immunoprecipitation of the NPT II protein, was repressed in the presence of cysteine and derepressed in its absence. Several fla- mutants were also isolated by Tn5-VB32 mutagenesis and shown to confer kanamycin resistance. Insertions within temporally regulated genes, such as those involved in flagellar biosynthesis and chemotaxis functions, can now be used directly to monitor transcriptional regulation from Caulobacter promoter sequences.

Bacteria↗
PubMed1984

Analysis of the pleiotropic regulation of flagellar and chemotaxis gene expression in Caulobacter crescentus by using plasmid complementation.

The biosynthesis of the single polar flagellum and the proteins that comprise the chemotaxis methylation machinery are both temporally and spacially regulated during the Caulobacter crescentus cell-division cycle. The genes involved in these processes are widely separated on the chromosome. The region of the chromosome defined by flaE mutations contains at least one flagellin structural gene and appears to regulate flagellin synthesis and flagellar assembly. The protein product of the adjacent flaY gene was found to be required to regulate the expression of several flagellin proteins and the assembly of a functional flagellum. We demonstrate here that each of these genes is also required for the expression of chemotaxis methylation genes known to map elsewhere on the chromosome. In order to study the regulation of these genes, plasmids were constructed that contain either an intact flaYE region or deletions in the region of flaY. These plasmids were mated into a wild-type strain and into strains containing various Tn5 insertion and deletion mutations and a temperature-sensitive mutation in the flaYE region. The presence of a plasmid containing the flaYE region allowed the mutant strains to swim and to exhibit chemotaxis, to synthesize increased amounts of the flagellins, to methylate their "methyl-accepting chemotaxis proteins" (MCPs), and to regain wild-type levels of methyltransferase activity. Chromosomal deletions that extend beyond the cloned region were not complemented by this plasmid. Plasmids containing small deletions in the flaY region failed to restore to any flaY or flaE mutants the ability to swim or to assemble a flagellar filament. When mated into a wild-type strain, plasmids bearing deletions in the flaY region were found to be recessive. The pleiotropic regulation of flagellin synthesis, assembly, and chemotaxis methylation functions exhibited by both the flaY and flaE genes suggest that their gene products function in a regulatory hierarchy that controls both flagellar and chemotaxis gene expression.

Bacteria↗
PubMed1984

Isolation and genetic analysis of Caulobacter mutants defective in cell shape and membrane lipid synthesis.

In this paper we report the isolation, characterization and genetic analysis of several C. crescentus mutants altered in membrane lipid synthesis. One of these, a fatty acid bradytroph, AE6002, was shown to be due to a mutation in the fatA gene. In addition to the presence of the fatA506 mutation, this strain was found to contain two other mutations, one of which caused the production of a water-soluble brown-orange pigment (pigA) and another which caused formation of helical cells (hclA). Expression of the latter two phenotypes required complex media and both were repressed by glucose. However, the lesions were mapped to loci that are separated by a substantial distance. The hclA and the fatA genes mapped close together, possibly implying that comutation had occurred in AE6002. Data are presented that allow the unambiguous identification of a second Fat gene (fatB) in C. crescentus. The map position of another mutation in membrane lipid biogenesis, the glycerol-3-PO4 auxotroph gpsA505, was also determined. During this study the flaZ gene was fine-mapped and the positions of proC and rif changed from the previously reported location.

Chromosome Mapping↗
PubMed1984

The windblown hip syndrome in total body cerebral palsy.

Windblown hips in patients with cerebral palsy are difficult to treat and predispose to poor, unstable sitting. In an attempt to identify the temporal sequence between dislocation of the hip, scoliosis, and pelvic obliquity, an in-depth clinical and radiological review of 22 teenage children was undertaken. The most common temporal sequence was dislocation of the hip, followed by pelvic obliquity, and finally scoliosis. It is recommended that the hip be closely monitored in infancy and that an aggressive treatment approach be undertaken if hip subluxation occurs. This is greatly facilitated by a good orthotic, therapy, and seating program to maintain the hips in the correct position. The maintenance of hip stability will facilitate seating as well as minimize the effects of the windblown hip syndrome.

Adolescent↗
PubMed1984

Caulobacter crescentus fatty acid-dependent cell cycle mutant.

A fatty acid auxotroph of Caulobacter crescentus, AE6001, which displays a strict requirement for unsaturated fatty acids to grow on glucose as the carbon source has been isolated. Starvation of AE6001 for unsaturated fatty acids resulted in a block in the cell cycle. Starved cultures accumulated at the predivisional cell stage after a round of DNA replication had been completed and after a flagellum had been assembled at the pole of the cell. Cell division and cell growth failed to occur probably because the mutant was unable to synthesize a membrane. An analysis of double mutants containing the fatB503 allele and other mutations in membrane biogenesis demonstrated that the cell cycle of AE6001 blocked at a homeostatic state. The addition of oleic acid to starved cultures permitted cell division and the initiation of a new round of DNA replication. The coincident block in both the initiation of DNA replication and membrane assembly, exhibited by starved cultures of this mutant, suggests that the fatB503 gene product may be involved in the coordination of these events.

Bacteria↗
PubMed1984

Genetic analysis and characterization of a Caulobacter crescentus mutant defective in membrane biogenesis.

A mutant of Caulobacter crescentus has been isolated which has an auxotrophic requirement for unsaturated fatty acids or biotin for growth on medium containing glucose as the carbon source. This mutant exhibits a pleiotropic phenotype which includes (i) the auxotrophic requirement, (ii) cell death in cultures attempting to grow on glucose in the absence of fatty acids or biotin, and (iii) a major change in the outer membrane protein composition before cell death. This genetic lesion did not appear to affect directly a fatty acid biosynthetic reaction because fatty acid and phospholipid syntheses were found to continue in the absence of supplement. Oleic acid repressed fatty acid biosynthesis and induced fatty acid degradation in the wild-type parent, AE5000 . The mutant strain, AE6000 , was altered in both of these regulatory functions. The AE6000 mutant also showed specific inhibition of the synthesis of outer membrane and flagellar proteins. Total phospholipid, DNA, RNA, and protein syntheses were unaffected. The multiple phenotypes of the AE6000 mutant were found to cosegregate and to map between hclA and lacA on the C. crescentus chromosome. The defect in this mutant appears to be associated with a regulatory function in membrane biogenesis and provides evidence for a direct coordination of membrane protein synthesis and lipid metabolism in C. crescentus.

Bacteria↗
PubMed1984

Chronic haemolytic anaemia in hypertrophic cardiomyopathy.

A patient with hypertrophic cardiomyopathy was found to have a chronic intravascular haemolytic anaemia. After investigation this was attributed to the abnormal haemodynamics of the cardiac disease. Treatment with propranolol was associated with an improvement in the anaemia. No further cases of haemolytic anaemia were found in 37 other patients with hypertrophic cardiomyopathy. It is concluded that haemolytic anaemia is a very rare feature of the condition.

Adult↗
PubMed1983

Phospholipase A2 modulation by calmodulin, prostaglandins and cyclic nucleotides.

Phospholipase A2 in the presence of Ca2+ was stimulated by calmodulin and by prostaglandin F2 alpha. Prostaglandin E2, cyclic-AMP and cyclic-GMP inhibited phospholipase A2 in the presence or absence of calmodulin. Dimethylsuberimidate cross-linking of phospholipase A2 with calmodulin was found to be Ca2+ dependent. These results indicate that phospholipase A2 is directly regulated by a host of key intracellular regulators and is one of the calmodulin-regulated enzymes.

Animals↗
PubMed1983

Differential template recognition by the Caulobacter crescentus and the escherichia coli RNA polymerases.

Transcription of Escherichia coli and Caulobacter crescentus phage DNAs by their respective host RNA polymerase was examined to determine their ability to recognize specific transcription signals on the heterologous template. Analysis of coliphage T7 in vitro transcripts showed that, like the E. coli enzyme, the C. crescentus RNA polymerase initiated transcription from the three major T7 early promoters and recognized the terminator at the end of the early region. On the other hand, several differences were found between the C. crescentus and E. coli RNA polymerases with respect to their interaction with Caulobacter phage phiCdl DNA. The rates of open complex formation and RNA elongation were slower when phiCdl DNA was transcribed by the E. coli RNA polymerase. In addition, transcription of phiCdl DNA by the E. coli enzyme produced a subset of transcripts not synthesized by the C. crescentus enzyme. The production of these different transcripts by the E. coli enzyme was dependent on salt concentration and, in at least one case, appeared to be the result of differential termination. Although both enzymes protected the same sites on phiCdl DNA from cleavage with HincII, the E. coli enzyme was unable to form stable complexes with some phiCdl restriction fragments that formed stable complexes with the C. crescentus RNA polymerase. These results indicate that although the C. crescentus RNA polymerase can accurately recognize transcription signals on a heterologous phage template, the E. coli enzyme exhibits altered specificity with a heterologous phage template of higher G + C content.

Bacteriophages↗
PubMed1983

Purification and characterization of an RNA processing enzyme from Caulobacter crescentus.

An RNA processing enzyme has been isolated from Caulobacter crescentus which is specific for double-stranded RNA, has an absolute requirement for monovalent cations, and can be eluted from a poly I:C agarose affinity column in pure form. This enzyme, like RNase III isolated from Escherichia coli, processes precursor ribosomal RNAs and polycistronic phage mRNAs and has a monomeric Mr of approximately 20,000. The two enzymes differ, however, in the recognition of specific cleavage sites and yield different digestion products when either coliphage T7 or C. crescentus phage phi Cdl early mRNA is used as substrate. Two lines of evidence are presented which show that an RNase III activity functions as a processing enzyme in C. crescentus. (a) In an in vitro reaction, C. crescentus phage phi Cdl major early mRNA synthesized in vitro by host RNA polymerase was processed by RNase III to yield RNA species which co-migrated with phage RNA synthesized in vivo in phi Cdl-infected cells, and (b) an in vitro transcript of a C. crescentus DNA clone containing the entire 16 S gene and part of the 23 S gene was processed by C. crescentus RNase III to yield an RNA product which co-migrated with 16 S RNA. The RNase III activity isolated from C. crescentus cell extracts has potential use in the analysis of specific RNA species because it was found to be more stringent in the recognition of cleavage sites than the E. coli enzyme.

Cloning, Molecular↗
PubMed1983

Vulvitis circumscripta plasmacellularis.

In this paper we present what is apparently the tenth published case of vulvitis circumscripta plasmacellularis (Zoon). The patient had eroded, red-brown, asymptomatic vulvar lesions that were recalcitrant to treatment. The characteristic histopathology--dense plasma cell infiltrate and deposition of hemosiderin--was present.

Adult↗
PubMed1983

Methylation involved in chemotaxis is regulated during Caulobacter differentiation.

Caulobacter crescentus carries a flagellum and is motile only during a limited time in its cell cycle. We have asked if the biochemical machinery that mediates chemotaxis exists coincident with the cell's structural ability to respond to a chemotactic signal. We first demonstrated that one function of the chemotaxis machinery, the ability to methylate the carboxyl side chains of a specific set of membrane proteins (methyl-accepting chemotaxis proteins, MCPs), is present in C. crescentus. This conclusion is based on the observations that (i) methionine auxotrophs starved of methionine can swim only in the forward direction (comparable to smooth swimming in the enteric bacteria), (ii) a specific set of membrane proteins was found to be methylated in vivo and the incorporated [3H]methyl groups were alkali sensitive, (iii) this same set of membrane proteins incorporated methyl groups from S-adenosylmethionine in vitro, and (iv) out of a total of eight generally nonchemotactic mutants, two were found to swim only in a forward direction and one of these lacked methyltransferase activity. Analysis of in vivo and in vitro methylation in synchronized cultures showed that the methylation reaction is lost when the flagellated swarmer cell differentiates into a stalked cell. In vivo methylation reappeared coincident with the biogenesis of the flagellum just prior to cell division. In vitro reconstitution experiments with heterologous cell fractions from different cell types showed that swarmer cells contain methyltransferase and their membranes can be methylated. However, newly differentiated stalked cells lack methyltransferase activity and membranes from these cells cannot accept methyl groups. These results demonstrate that MCP methylation is confined to that portion of the cell cycle when flagella are present.

Bacteria↗