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L Shapiro

Publications and source records attributed to L Shapiro.

At least 19 recordsLinked to original sources
PubMed1992

Organization and ordered expression of Caulobacter genes encoding flagellar basal body rod and ring proteins.

The biogenesis of the polar flagellum in Caulobacter crescentus is limited to a specific time in the cell cycle and to a specific site on the cell. The basal body is the first part of the flagellum to be assembled. In this report we identify a cluster of genes encoding basal body components and describe their transcriptional regulation. The genes in this cluster form an operon whose expression is controlled temporally. The first two genes encode homologs of FlgF and FlgG, which are the proximal and distal rod proteins, respectively. The sequences of the N and C termini of the Salmonella typhimurium flagellar axial proteins, rod, hook and HAP-1, known to be highly conserved, share a high degree of sequence identity with the FlgF and FlgG rod proteins of the distantly related, C. crescentus. Two additional genes in the flgF, flgG operon, flaD and flgH, both encode proteins with potentially cleavable signal sequences. The flgH gene, encoding the L-ring protein, is also transcribed from an internal promoter. Transcription from the flgF promoter initiates prior to initiation at the internal flgH promoter. The internal promoter and its activator site reside within the C-terminal coding sequence of the upstream flaD gene. This type of gene overlap is also observed in bacterial genes involved in cell division. Flagellum biogenesis, like cell division, is a morphogenic event that requires the orderly assembly of component proteins and the overlapping gene organization may affect this "ordering" of assembly. The promoters for the flgF operon and the flgH gene use sigma 54 to initiate transcription. The use of sigma 54 promoters, known to require cognate binding proteins, could allow the fine-tuning that provides the temporal ordering of flagellar gene transcription. In this context, we have found that the flgF operon and the distal flgI gene encoding the P-ring, share a sigma 54 activator sequence (class IIA) that differs from the flgH L-ring gene sigma 54 activator site (class IIB) and the hook cluster (class IIC) sigma 54 activator site. The sequential activation of these three subgroups of structural genes reflects the order of assembly of their gene products into the flagellum.

Amino Acid Sequence↗
PubMed1992

Cell-cycle control of a cloned chromosomal origin of replication from Caulobacter crescentus.

Caulobacter crescentus cell division is asymmetric and yields distinct swarmer cell and stalked cell progeny. Only the stalked cell initiates chromosomal replication, and the swarmer cell must differentiate into a stalked cell before chromosomal DNA replication can occur. In an effort to understand this developmental control of replication, we employed pulsed-field gel electrophoresis to localize and to isolate the chromosomal origin of replication. The C. crescentus homologues of several Escherichia coli genes are adjacent to the origin in the physical order hemE, origin, dnaA and dnaK,J. Deletion analysis reveals that the minimal sequence requirement for autonomous replication is greater than 430 base-pairs, but less than 720 base-pairs. A plasmid, whose replication relies only on DNA from the C. crescentus origin of replication, has a distinct temporal pattern of DNA synthesis that resembles that of the bona fide C. crescentus chromosome. This implies that cis-acting replication control elements are closely linked to this origin of replication. This DNA contains sequence motifs that are common to other bacterial origins, such as five DnaA boxes, an E. coli-like 13-mer, and an exceptional A + T-rich region. Point mutations in one of the DnaA boxes abolish replication in C. crescentus. This origin also possesses three additional motifs that are unique to the C. crescentus origin of replication: seven 8-mer (GGCCTTCC) motifs, nine 8-mer (AAGCCCGG) motifs, and five 9-mer (GTTAA-n7-TTAA) motifs are present. The latter two motifs are implicated in essential C. crescentus replication functions, because they are contained within specific deletions that abolish replication.

Bacterial Proteins↗
PubMed1992

Solution structure of a trinucleotide A-T-A bulge loop within a DNA duplex.

We have synthesized an oligodeoxynucleotide duplex, d(G-C-A-T-C-G-A-T-A-G-C-T-A-C-G).d(C-G-T-A-G-C-C-G-A-T-C-G), with a three-base bulge loop (A-T-A) at a central site in the first strand. Nuclear Overhauser experiments (NOESY) in H2O indicate that the GC base pairs flanking the bulge loop are intact between 0 and 25 degrees C. Nuclear Overhauser effects in both H2O and D2O indicate that all bases within the bulge loop are stacked into the helix. These unpaired bases retain an anti conformation about their glycosidic bonds as they stack within the duplex. The absence of normal sequential connectivities between the two cytosine residues flanking the bulge site on the opposite strand indicates a disruption in the geometry of this base step upon insertion of the bulged bases into the helix. This conformational perturbation is more akin to a shearing apart of the bases, which laterally separates the two halves of the molecule, rather than the "wedge" model often invoked for single-base bulges. Using molecular dynamics calculations, with both NOE-derived proton-proton distances and relaxation matrix-calculated NOESY cross peak volumes as restraints, we have determined the solution structure of an A-T-A bulge loop within a DNA duplex. The bulged bases are stacked among themselves and with the guanine bases on either side of the loop. All three of the bulged bases are displaced by 2-3 A into the major groove, increasing the solvent accessibility of these residues. The ATA-bulge duplex is significantly kinked at the site of the lesion, in agreement with previously reported electron microscopy and gel retardation studies on bulge-containing duplexes [Hsieh, C.-H., & Griffith, J. D. (1989) Proc. Natl. Acad. Sci. U.S.A 86, 4833-4837; Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840]. Bending occurs in a direction away from the bulge-containing strand, and we find a significant twist difference of 84 degrees between the two base pairs flanking the bulge loop site. This value represents 58% of the twist difference for base pairs four steps apart in B-DNA. These results suggest a structural mechanism for the bending of DNA induced by unpaired bases, as well as accounting for the effect bulge loops may have on the secondary and tertiary structures of nucleic acids.

Adenine↗
PubMed1992

Directed isolation of human genes that escape X inactivation.

Existing methodologies have been combined to produce a directed approach to the isolation of human genes that escape X inactivation. A mouse-human somatic cell hybrid line was established that has an inactive X as its only human chromosome, and nuclear RNA from this cell line was used to construct a cDNA library. Transcribed human sequences were isolated by screening the library with labeled human DNA. The corresponding genomic sequences were isolated in phage or cosmid clones, and exons were identified by detection of transcripts on northern blots. By these means three human loci have been identified that contain genes expressed from an inactive X chromosome. Fluorescence in situ hybridization has been used to map these genes to Xp21.1-22.1, Xp22.1-22.2, and terminal Xp/Yp. One of the three genes (XE45) corresponds to the ZFX gene, while the other two genes (XE7 and XE59) represent novel cloned sequences. Physical and genetic evidence indicate that XE7 is a newly identified pseudoautosomal gene.

Animals↗
PubMed1992

Isolation and characterization of a yeast artificial chromosome (YAC) contig around the human steroid sulfatase gene.

The region surrounding the steroid sulfatase (STS) locus on Xp22.3 is of particular interest since it represents a deletion hot spot, shares homology with the proximal long arm of the Y chromosome (Yq11.2), and contains genes for several well-described X-linked disorders. Here we describe yeast artificial chromosomes (YACs) covering 450 kb around the STS gene. Eight YAC clones were isolated from a human YAC library. Their STS exon content was determined and the overlap of the clones characterized. Two of the YAC clones were found to contain the entire STS gene. The most proximal and the most distal ends of the YAC contig were cloned but neither of them crossed the breakpoints in any of the previously described patients with entire STS gene deletions. This is consistent with deletions larger than 500 kb in all these patients. One of the YAC clones was found to contain sequences from the STS pseudogene on Yq11.2. Two anonymous DNA sequences, GMGXY19 and GMGXY3, previously mapped in the vicinity of the STS locus, were found within the YAC contig and their assignment with respect to the STS locus was thus possible. This contig is useful for the overlap cloning of the Xp22.3 region and for reverse genetic strategies for the isolation of disease genes in the region. Furthermore, it may provide insight into the molecular mechanisms of deletion and translocation events on Xp22.3 and in the evolution of sex chromosomes.

Arylsulfatases↗
PubMed1992

A developmentally regulated Caulobacter flagellar promoter is activated by 3' enhancer and IHF binding elements.

The transcription of a group of flagellar genes is temporally and spatially regulated during the Caulobacter crescentus cell cycle. These genes all share the same 5' cis-regulatory elements: a sigma 54 promoter, a binding site for integration host factor (IHF), and an enhancer sequence, known as the ftr element. We have partially purified the ftr-binding proteins, and we show that they require the same enhancer sequences for binding as are required for transcriptional activation. We have also partially purified the Caulobacter homolog of IHF and demonstrate that it can facilitate in vitro integrase-mediated lambda recombination. Using site-directed mutagenesis, we provide the first demonstration that natural enhancer sequences and IHF binding elements that reside 3' to the sigma 54 promoter of a bacterial gene, flaNQ, are required for transcription of the operon, in vivo. The IHF protein and the ftr-binding protein is primarily restricted to the predivisional cell, the cell type in which these promoters are transcribed. flaNQ promoter expression is localized to the swarmer pole of the predivisional cell, as are other flagellar promoters that possess these regulatory sequences 5' to the start site. The requirement for an IHF binding site and an ftr-enhancer element in spatially transcribed flagellar promoters indicates that a common mechanism may be responsible for both temporal and polar transcription.

Amino Acid Sequence↗
PubMed1992

Isolation of a new gene from the distal short arm of the human X chromosome that escapes X-inactivation.

A gene, designated GS1, was identified by its association with a CpG island approximately 100 kb telomeric to the steroid sulfatase (STS) locus on the distal short arm of the human X chromosome. Both cDNA and genomic clones of the GS1 gene have been isolated and characterized. The cDNA clone detects a 2.3 kb transcript in human placenta and fibroblasts, and may encode a protein of 214 amino acid residues. Although sequences homologous to GS1 cDNA are present on chromosomes 1, 20, X, and Y, the functional GS1 gene is on the X chromosome. The GS1 gene appears to be non-essential, as there are no obvious clinical differences between STS deficient patients with point mutations in the STS gene, and patients with a deletion of the STS and GS1 genes. The GS1 gene is expressed from mouse-human cell hybrids containing active or inactive human X chromosomes, indicating that it escapes X inactivation. Characterization of GS1 genomic clones revealed that the gene consists of 4 exons spanning over 105 kb, with its transcriptional direction opposite to that of the STS gene. The isolation and characterization of a new gene which escapes X inactivation from distal Xp is of interest as it adds to our understanding of the structural organization of the human X chromosome and may help in providing clues regarding the mechanism of X-inactivation.

Amino Acid Sequence↗
PubMed1992

A temporally controlled sigma-factor is required for polar morphogenesis and normal cell division in Caulobacter.

The transcription of many spatially and temporally controlled flagellar structural genes in Caulobacter requires the RNA polymerase sigma 54 subunit. Like flagellar biogenesis, stalk formation is an asymmetric polar morphogenesis that occurs once each cell cycle in response to internal cell cycle signals. We have isolated the sigma 54 gene (rpoN) and describe here a novel role for this alternative sigma-factor in cell differentiation: It is required for the biogenesis of both polar structures, and the disruption of the rpoN gene results in aberrant cell division. Surprisingly, the transcription of rpoN is temporally regulated during the cell cycle; it increases 10-fold commensurate with stalk formation and just before the onset of flagellar gene expression. These results suggest that sigma 54 abundance responds to cell cycle cues and is involved in the global timing of the central events of Caulobacter development, whereas the transcriptional activators of sigma 54-dependent promoters are responsible for the refined control of the expression of individual or small groups of genes required for each specific event.

Amino Acid Sequence↗
PubMed1992

Polar localization of a bacterial chemoreceptor.

The bacterial chemotaxis signal transducer MCP is an integral membrane receptor protein. The chemoreceptor is localized at the flagellum-bearing pole of Caulobacter crescentus swarmer cells. Amino-terminal sequences of the MCP target the protein to the membrane while the carboxy-terminal portion of the protein is responsible for polar localization. The C. crescentus and Escherichia coli MCPs have highly conserved carboxy-terminal domains, and when an E. coli MCP is expressed in C. crescentus, it is targeted to the swarmer cell progeny. These results suggest that subcellular localization of a prokaryotic protein involves interaction of specific regions of the protein with unique cell sites that contain either localized binding proteins or a specific secretory apparatus.

Amino Acid Sequence↗
PubMed1992

Early Caulobacter crescentus genes fliL and fliM are required for flagellar gene expression and normal cell division.

The biogenesis of the Caulobacter crescentus polar flagellum requires the expression of more than 48 genes, which are organized in a regulatory hierarchy. The flbO locus is near the top of the hierarchy, and consequently strains with mutations in this locus are nonmotile and lack the flagellar basal body complex. In addition to the motility phenotype, mutations in this locus also cause abnormal cell division. Complementing clones restore both motility and normal cell division. Sequence analysis of a complementing subclone revealed that this locus encodes at least two proteins that are homologs of the Salmonella typhimurium and Escherichia coli flagellar proteins FliL and FliM. FliM is thought to be a switch protein and to interface with the flagellum motor. The C. crescentus fliL and fliM genes form an operon that is expressed early in the cell cycle. Tn5 insertions in the fliM gene prevent the transcription of class II and class III flagellar genes, which are lower in the regulatory hierarchy. The start site of the fliLM operon lies 166 bp from the divergently transcribed flaCBD operon that encodes several basal body genes. Sequence comparison of the fliL transcription start site with those of other class I genes, flaS and flaO, revealed a highly conserved 29-bp sequence in a potential promoter region that differs from sigma 70, sigma 54, sigma 32, and sigma 28 promoter sequences, suggesting that at least three class I genes share a unique 5' regulatory region.

Amino Acid Sequence↗
PubMed1992

Expression of an early gene in the flagellar regulatory hierarchy is sensitive to an interruption in DNA replication.

Genes involved in the biogenesis of the flagellum in Caulobacter crescentus are expressed in a temporal order and are controlled by a trans-acting regulatory hierarchy. Strains with mutations in one of these genes, flaS, cannot transcribe flagellar structural genes and divide abnormally. This gene was cloned, and it was found that its transcription is initiated early in the cell cycle. Subclones that restored motility to FlaS mutants also restored normal cell division. Although transcription of flaS was not dependent on any other known gene in the flagellar hierarchy, it was autoregulated and subject to mild negative control by other genes at the same level of the hierarchy. An additional level of control was revealed when it was found that an interruption of DNA replication caused the inhibition of flaS transcription. The flaS transcript initiation site was identified, and an apparently unique promoter sequence was found to be highly conserved among the genes at the same level of the hierarchy. The flagellar genes with this conserved 5' region all initiate transcription early in the cell cycle and are all sensitive to a disruption in DNA replication. Mutations in these genes also cause an aberrant cell division phenotype. Therefore, flagellar genes at or near the top of the hierarchy may be controlled, in part, by a unique transcription factor and may be responsive to the same DNA replication cues that mediate other cell cycle events, such as cell division.

Base Sequence↗
PubMed1991

Sensitivity to triazolam in the elderly.

BACKGROUND: Elderly persons frequently appear to be sensitive to the effects of many drugs that depress the central nervous system. We studied the effect of age on the pharmacokinetics and pharmacodynamics of the benzodiazepine hypnotic agent triazolam, now the most frequently prescribed hypnotic drug in the United States. METHODS: Twenty-six healthy young subjects (mean age, 30 years) and 21 healthy elderly subjects (mean age, 69 years) participated in a four-way crossover study. After a single-blind adaptation trial with placebo, each subject received, in random order and in double-blind fashion, single doses of placebo, 0.125 mg of triazolam, and 0.25 mg of triazolam. For 24 hours after the administration of each of the three study medications, plasma triazolam levels were determined and psychomotor performance, memory, and degree of sedation were assessed. RESULTS: Plasma triazolam concentrations increased in proportion to the dose, but the elderly subjects had higher plasma concentrations due to reduced clearance of the drug. The degree of sedation as rated by an observer and the reduction in the subjects' performance on the digit-symbol substitution test were both greater in the elderly than in the young subjects after they were given the same doses. The relation of the plasma triazolam concentration to the degree of impairment was similar for the two groups. As part of the study, information was presented 1 1/2 hours after the administration of the drugs; the subjects' ability to recall the information 24 hours later was impaired by both doses of triazolam, and the percent decrease was similar in the young and elderly groups. CONCLUSIONS: Triazolam caused a greater degree of sedation and greater impairment of psychomotor performance in healthy elderly persons than in young persons who received the same dose. These effects resulted from reduced clearance and higher plasma concentrations of triazolam rather than from an increased intrinsic sensitivity to the drug. On the basis of these results, the dosage of triazolam for elderly persons should be reduced on average by 50 percent.

Adult↗
PubMed1991

Expression of positional information during cell differentiation of Caulobacter.

The asymmetric targeting of proteins to the Caulobacter predivisional cell poles yields dissimilar progeny. We show that the products of transcriptional reporter gene fusions to a flagellin gene and to the flagellar hook operon are segregated to the progeny swarmer cell. This segregation does not depend on sequences within the mRNA, but on the upstream regulatory region. The subset of developmentally regulated flagellar genes that exhibit mRNA segregation has the same upstream cis-acting elements: an activator-binding site known as the ftr sequence and an IHF-binding site. We propose that these genes are preferentially transcribed from the chromosome in the incipient swarmer cell pole of the predivisional cell.

Bacteria↗
PubMed1991

Identification of cis and trans-elements involved in the timed control of a Caulobacter flagellar gene.

The genes encoding the structural components of the Caulobacter crescentus flagellum are temporally controlled and their order of expression reflects the sequence of assembly. Transcription of the operon containing the structural gene for the flagellar hook protein occurs at a defined time in the cell cycle, and information necessary for transcription is contained within a region between -81 and -120 base-pairs from the transcription start site. To identify the sequence elements that contribute to the temporal control of hook operon transcription, we constructed deletions and base changes in the 5' region and fused the mutagenized regulatory region to transcription reporter genes. We demonstrate that sequences 3' to the transcription start site do not contribute to temporal control. We confirm that upstream sequences between -81 and -120 base-pairs are necessary for temporal activation, and that transcription also requires sequences at -26 to -46 base-pairs. A specific binding activity for the region between -81 and -122 base-pairs was shown to be temporally controlled, appearing prior to the activation of hook operon transcription. This binding activity was missing from strains containing mutations in flaO and flaW, two genes near the top of the flagellar hierarchy known to be required for hook operon transcription. Thus, the hook operon upstream region contains a sequence element that responds to a temporally controlled trans-acting factor(s), and in concert with a second sequence element causes the timed activation of transcription.

Bacterial Proteins↗
PubMed1991

Real-time examinations of lexical processing in aphasics.

We argue that the lesion localizing value of disruptions to modular information processing systems emerges most clearly from on-line analyses of processing. In this respect we seek to show that left anterior (but not left posterior) damage causes slowed information access and we discuss the manner in which this slowing might yield some of the specific syntactic limitations charted in Broca's aphasia. The general possibility we raise is that the cortical area implicated in Broca's aphasia is not necessarily the locus of syntactic representations, but rather sustains particular time-based operating characteristics that in turn sustain normal real-time parsing.

Aphasia, Broca↗
PubMed1991

Positional information during Caulobacter cell differentiation.

The formation of two distinct daughter cells upon division of the bacterium Caulobacter crescentus is the result of asymmetry in the predivisional cell, in part due to localization of both flagellar and chemotaxis proteins to the swarmer cell pole. Recent evidence suggests that both localized transcription and protein targeting directed by specific amino acid sequence are involved in the localization.

Bacterial Proteins↗
PubMed1991

Genetic analysis of a temporally transcribed chemotaxis gene cluster in Caulobacter crescentus.

Caulobacter crescentus performs chemotaxis by short intermittent reversals of rotation of its single polar flagellum. Tn5 insertions causing a general chemotaxis phenotype, an inability to reverse swimming direction and to form large swarm colonies, have been mapped to an 8-kb region of the C. crescentus genome. These Tn5 mutations had different effects on the methyl-accepting chemotaxis proteins (MCP), and the activities of methyltransferase and methylesterase. The Tn5 insertion mutant SC1130 had no cross-reacting MCP and had reduced levels of activity of the methyltransferase and methylesterase. Other mutants bearing Tn5 insertions retained cross-reacting MCP activity and were altered only in their methyltransferase and methylesterase activities. Using a cosmid library we isolated a clone that complemented SC1130. Complementation studies of the Tn5 mutants using derivatives of the cosmid clone showed that all the Tn5 insertions lie within a single operon that appears to encode many chemotaxis genes. The first gene in this operon was shown to encode an MCP by immuno-blot analysis of strains carrying beta-galactosidase protein fusions to portions of the operon. The promoter of this operon was located by chromosomal integration of subclones of this region and by identifying DNA fragments that were capable of expressing lacZ transcriptional fusions. The transcription of the che operon occurred at a defined time in the cell cycle, prior to cell division.

Bacterial Proteins↗