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Biomedical subjects

D Court

Publications and source records attributed to D Court.

At least 19 recordsLinked to original sources
PubMed2001

Cell toxicity caused by products of the p(L) operon of bacteriophage lambda.

Induction of a lambda prophage causes the death of the host cell even in the absence of phage replication and lytic functions due to expression of functions from the lambda p(L) operon. We genetically modified the lambda prophage to determine which lambda p(L) operon functions were involved in cell killing. Viability assays and flow cytometry were used to monitor cell death and filamentation. The kil gene was shown to cause cell death and filamentation as described previously. Another killing activity was mapped within the p(L) operon to the gam gene. Inspection of the DNA sequence showed that there are two possible translation start points for both kil and gam. In both cases, the shorter of the two possible products could cause cell killing. The shorter products were also sufficient for the known filamentation and recombination activities of the respective Kil and Gam functions. The expression level of the p(L) operon is down-regulated by Cro repressor. In the absence of Cro, higher p(L) expression levels allow either Kil or Gam to be lethal or growth inhibitory, whereas at lowered expression in Cro-repressed conditions, only Kil is lethal. The filamentation function of Kil and recombination activity of Gam are unaffected at Cro-repressed levels of expression.

Bacterial Proteins↗
PubMed1999

Cloning and analysis of the rnc-era-recO operon from Pseudomonas aeruginosa.

The rnc operon from Pseudomonas aeruginosa has been cloned and characterized. The three genes comprising this operon, rnc, era, and recO, are arranged similarly to those in some other gram-negative bacteria. Multicopy plasmids carrying the rnc operon of P. aeruginosa functionally complement mutations of the rnc, era, and recO genes in Escherichia coli. In particular, the P. aeruginosa era homolog rescues the conditional lethality of era mutants in E. coli, and the presumptive protein has 60% identity with the Era of E. coli. We discuss these data and evidence suggesting that a GTPase previously purified from P. aeruginosa and designated Pra is not an Era homolog.

Bacterial Proteins↗
PubMed1997

A c-erbB-2 promoter-specific nuclear matrix protein from human breast tumor tissues mediates NF-kappaB DNA binding activity.

The c-erbB-2 gene overexpression plays a major role in the pathogenesis of breast cancer. Binding studies detected a nuclear matrix protein (NMP) in human breast tumor tissues that recognizes a matrix attachment region (MAR) in the immediate vicinity of the c-erbB-2 gene promoter. This NMP is expressed in breast tumor tissues and cell lines along with c-erbB-2, but is not found in corresponding normal tissues. Furthermore, when NMP purified from the breast tumors by its affinity to the MAR sequence is added to nuclear extracts of breast cancer cells, it selectively stimulates the binding of the NF-kappaB transcription factor to DNA. A model is suggested in which the association of the MAR-like sequence with the nuclear matrix raises the local concentration of the specific NMP, which in turn interacts with the nuclear factor NF-kappaB to increase its local level. Such a complex could explain at a molecular level the "increase in NF-kappaB DNA binding activity" often observed in c-erbB-2- and BRCA1-positive human breast tumors. The increased NF-kappaB activity could thereby contribute to breast cancer progression.

Antigens, Nuclear↗
PubMed1996

Point mutations in a transcription terminator, lambda tI, that affect both transcription termination and RNA stability.

The terminator tI is located approx. 280 nucleotides beyond the int gene of bacteriophage lambda. Besides its role as a transcription terminator, tI may confer stability to the int message by protecting it from 3' exonucleolytic degradation. In order to study the role of the tI sequence in transcription termination and RNA stability, three different point mutations tI1, tI2, and tI3 were isolated and characterized. All the tI mutations map in the G + C-rich region of dyad symmetry in the terminator and decrease the transcriptional termination of tI in vivo from 99% for the wild type terminator to 81-93% as determined by galactokinase activity and in vitro from 80% for the wild type terminator to 8-12% using the E. coli RNA polymerase. Additionally, the tI mutations cause upstream transcript instability in vivo. This instability defect caused by tI mutations is compensated by the host mutant deficient in polynucleotide phosphorylase resulting in increased steady state levels of these mutant transcripts. The results show that the intact hairpin of tI is essential for efficient transcription termination and for maintaining mRNA stability by blocking the 3' to 5' exonucleolytic activity of polynucleotide phosphorylase.

Bacteriophage lambda↗
PubMed1996

A nuclear matrix-specific factor that binds a specific segment of the negative regulatory element (NRE) of HIV-1 LTR and inhibits NF-kappa(B) activity.

The negative regulatory element (NRE) of human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR) is a defined region that has been reported to downregulate LTR-directed HIV gene expression. However, information on the precise role of this region in regulating HIV gone transcription is lacking. We have investigated the possibility that these NRE sequences regulate HIV transcription by a mechanism mediated through a nuclear matrix-specific DNA-protein interaction. We find a nuclear matrix attachment region (MAR) present within the NRE of the HIV-1 LTR that recognizes a sequence-specific DNA-binding protein present in the nuclear matrix of HIV infected cells. Moreover, we also show that the purified DNA-binding nuclear matrix protein (NMP) specifically represses the DNA-binding activity of NF-kappaB. It is likely that the MAR and MAR-enriched specific DNA-binding NMP are brought into juxtaposition by the non-chromatin scaffolding of the nucleus, thus influencing NF-kappaB (and other nuclear proteins) DNA-binding activity through protein-protein and protein-DNA interactions. Our date suggest that one possible role of the NRE could be to act as a matrix attachment site in the nuclear matrix, thus, allowing interaction with a sequence-specific trans-acting factor. The negative effect on NF-kappaB activity due to this MAR-NMP-specific interaction provides a mechanism by which the NRE downregulates HIV gene expression.

Antigens, Nuclear↗
PubMed1995

Human gamma X satellite DNA: an X chromosome specific centromeric DNA sequence.

The cosmid clone, CX16-2D12, was previously localized to the centromeric region of the human X chromosome and shown to lack human X-specific alpha satellite DNA. A 1.2 kb EcoRI fragment was subcloned from the CX16-2D12 cosmid and was named 2D12/E2. DNA sequencing revealed that this 1,205 bp fragment consisted of approximately five tandemly repeated DNA monomers of 220 bp. DNA sequence homology between the monomers of 2D12/E2 ranged from 72.8% to 78.6%. Interestingly, DNA sequence analysis of the 2D12/E2 clone displayed a change in monomer unit orientation between nucleotide positions 585-586 from a "tail-to-head" arrangement to a "head-to-tail" configuration. This may reflect the existence of at least one inversion within this repetitive DNA array in the centromeric region of the human X chromosome. The DNA consensus sequence derived from a compilation of these 220 bp monomers had approximately 62% DNA sequence similarity to the previously determined gamma 8 satellite DNA consensus sequence. Comparison of the 2D12/E2 and gamma 8 consensus sequences revealed a 20 bp DNA sequence that was well conserved in both DNA consensus sequences. Slot-blot analysis revealed that this repetitive DNA sequence comprises approximately 0.015% of the human genome, similar to that found with gamma 8 satellite DNA. These observations suggest that this satellite DNA clone is derived from a subfamily of gamma satellite DNA and is thus designated gamma X satellite DNA. When genomic DNA from six unrelated males and two unrelated females was cut with SstI or HpaI and separated by pulsed-field gel electrophoresis, no restriction fragment length polymorphisms were observed for either gamma X (2D12/E2) or gamma 8 (50E4) probes. Fluorescence in situ hybridization localized the 2D12/E2 clone to the lateral sides of the primary constriction specifically on the human X chromosome.

Base Sequence↗
PubMed1993

Cloning and organization of the abc and mdl genes of Escherichia coli: relationship to eukaryotic multidrug resistance.

Using degenerate oligodeoxyribonucleotides from conserved regions of the gene family encoding ATP-binding domain of the active transporter, two new Escherichia coli genes were identified. The first of the genes, named mdl (multidrug resistance-like), is located at min 10.2 of the E. coli chromosome and encodes two ATP-binding motifs and two hydrophobic (transmembrane) domains. The ATP-binding domains of mdl show 35-38% amino acid (aa) identity with members of the eukaryotic P-glycoprotein/multidrug resistance family. To date, 25 members of the ATP-transporter/permease gene family have been characterized in E. coli. Comparison of the ATP-binding domains from this family indicates that mdl is part of a distinct subfamily of sequences that includes hlyB, msbA, and cvaB. Gene-disruption studies revealed that mdl is not essential for cell growth. The second open reading frame, named abc (ATP-binding cassette), is located at min 4.9 of the chromosome, encodes a single ATP-binding domain, and is most homologous to ftsE, a cell division control gene of E. coli. The abc gene product also shows aa sequence homology to several E. coli permeases.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Isolation and identification of a novel tandemly repeated DNA sequence in the centromeric region of human chromosome 8.

EcoRI subclones, designated as 50E1 and 50E4, were independently obtained from a cosmid clone previously mapped to the centromeric region of human chromosome 8. Southern blot hybridization analyses suggested that both subclones contain repetitive DNA sequences different from the chromosome 8 specific alphoid DNA. DNA sequence analysis of the 704 bp insert of 50E1 and the 1,962 bp insert of 50E4 revealed that both inserts contained tandemly repeated units of approximately 220 bp. Fluorescence in situ hybridization studies confirmed these two subclones to be specifically located on the centromeric region of chromosome 8. A 220 bp consensus sequence, derived from nine monomeric repeats, showed no significant homology to alphoid consensus sequences or to other currently known human centromeric DNA sequence. Furthermore, no significant homology was found with any other DNA sequence deposited in the EMBL or GenBank databases, indicating that this chromosome 8 specific repetitive DNA sequence is novel. From slot blot experiments it was estimated that 0.013% of the human genome comprises 1,750 of these monomeric repeats, residing on the centromeric region of chromosome 8 in tandem array(s).

Base Sequence↗
PubMed1992

Mapping of 50 cosmid clones isolated from a flow-sorted human X chromosome library by fluorescence in situ hybridization.

Fifty cosmids have been mapped to metaphase chromosomes by fluorescence in situ hybridization under conditions that suppress signals from repetitive DNA sequences. The cosmid clones were isolated from a flow-sorted human X chromosome library. Thirty-eight of the clones were localized to chromosome X and 12 to autosomes such as chromosomes 3, 7, 8, 14, and 17. Although most of the cosmids mapped to the X chromosome appeared to be scattered along both the short and long arms, 10 cosmids were localized to the centromeric region of the chromosome. Southern blot analysis revealed that only two of these clones hybridized to probe pXBR-1, which detects the DXZ1 locus. In addition, 4 out of 5 cosmids mapped on chromosome 8 also localized on the centromeric region. While localization of X-specific cosmids will facilitate the physical mapping of the human X chromosome, cosmids mapped to the centromeric regions of chromosomes X and 8 should be especially useful for studying the structure and organization of these regions.

Blotting, Southern↗
PubMed1990

Analysis of mutations in the ninR region of bacteriophage lambda that bypass a requirement for lambda N antitermination.

Two mutations in the ninR region of bacteriophage lambda that bypass a requirement for antitermination have been studied. One mutation, byp, has been cloned and mapped by marker rescue to a 417-base-pair segment in the ninR region of the genome. Analysis of the byp mutation by using promoter detection vectors, DNA sequencing, and S1 nuclease analysis showed that the byp mutation created a new promoter that transcribed gene Q. The second mutation analyzed was the deletion nin3. Sequence analysis revealed that 2,485 base pairs of the ninR region were removed, beginning within the ren gene and ending in an open reading frame termed ninG. The tR2 and tR3 terminators, and probably others, were removed by the nin3 deletion, thereby allowing the phage to be N independent and to grow in hosts defective for Nus antitermination factors.

Bacteriophage lambda↗
PubMed1987

Induction of the heat shock response of E. coli through stabilization of sigma 32 by the phage lambda cIII protein.

The cIII protein of phage lambda favors the lysogenic response to infection by inhibiting the degradation of the lambda cII protein, which exerts the primary control on the developmental decision for lysis or lysogeny. To study the mechanism and scope of cIII-mediated regulation, we have used plasmid systems to examine the specific effect of cIII overproduction on the growth of Escherichia coli and the synthesis of bacterial proteins. We have found that maximal production of cIII prolongs the heat-induced synthesis of E. coli heat shock proteins and provokes elevated production of heat shock proteins even at low temperature. The overproduction of heat shock proteins is correlated with a rapid inhibition of cell growth, as judged by measurements of optical density. We suggest that an overactive heat shock response inhibits bacterial growth, either because excessive production of one or more of the proteins is highly deleterious or because only heat shock promoters are transcribed efficiently. To examine the effect of cIII on sigma 32, the specificity factor for the heat shock response, we have studied the stability of sigma 32 in cells carrying both cIII- and sigma 32-producing plasmids; the half-life of sigma 32 is increased fourfold in the presence of cIII. We conclude that overproduction of cIII provokes the heat shock response by increasing the steady-state level of active sigma 32. These studies also support the concept that the rate of expression of heat shock proteins is directly correlated with the amount of active sigma 32 and that regulation of the stability of sigma 32 may be an important factor for control of the heat shock response.

Bacteriophage lambda↗
PubMed1987

The economic realities of the Health Service.

The Unit General Manager may normally be treated with total indifference or acute suspicion but anything he has to say about "efficiency savings" will certainly be treated with scepticism usually reserved for those claiming "the cheque is in the post". Staff will recognise this as another term for "cut backs". It may be most honest to say that since there is not enough money to cover increased demands it will have to be redistributed and this cannot be done without causing some suffering.

Financial Management↗
PubMed1986

Repression of a mutant derivative of the pRE promoter of bacteriophage lambda by its activator, CII.

A 2-bp insertion between the -10 and -35 regions of the pRE promoter of bacteriophage lambda reverses the effect of the activator protein, CII, on transcription from pRE in vitro. The mutant promoter is weakly constitutive in the absence of cII protein and repressed in its presence. This is in sharp contrast to wild-type pRE which is inactive in the absence of cII protein and stimulated at least 1000-fold in its presence (Shih and Gussin, 1984a; McClure and Hoopes, 1985). These effects are explained by the creation of a new -35 region with weak homology to the -35 consensus sequence for Escherichia coli promoters, and by the altered spatial relationship between the -35 region and the CII-binding site. This interpretation was confirmed by analysis of double mutants containing known cy (pRE) mutations together with the 2-bp insertion. Insertion of 4 bp or deletion of 2 bp completely inactivates pRE in the presence or absence of cII protein, again indicating that activation is dependent upon proper spacing between the -35 region and the transcription start point.

Bacteriophage lambda↗
PubMed1984

Removal of a terminator structure by RNA processing regulates int gene expression.

The int gene of phage lambda encodes a protein involved in site-specific recombination. Its expression is regulated differentially during successive phases of the lambda infective cycle. The gene is transcribed early after infection from one promoter, pL, and later from a second promoter pI. Each transcription event requires different positive activation factors, lambda N and cII proteins, respectively. Transcription from the pI promoter, located adjacent to int, passes through int and terminates 277 nucleotides beyond int at tI. Polymerases initiating at pL transcribe through tI and into the b segment of lambda DNA. The read-through pL transcript is sensitive to cleavage by the endonuclease, RNase III, both in vivo and in vitro. Two specific cuts are made by RNase III in a double-stranded structure about 260 nucleotides beyond int in the location of the tI terminator. Functionally, the processed pL transcript is unable to synthesize the int gene product, whereas the terminated and unprocessed pI transcript expresses int. Interestingly, unprocessed pL transcripts made in hosts defective in RNase III (rnc-) can express int. Thus a correlation exists between processing and negative control of int expression. The place where processing occurs, some 260 nucleotides beyond int, is called sib, and the control of int expression from this site is called retroregulation. Retroregulation by sib is not restricted just to the int gene; we show that if the sib site is cloned beyond a bacterial gene, the gene is controlled by sib and RNase III. Specific models are discussed with respect to control of gene expression by RNase III from a site beyond the controlled gene.

Bacteriophage lambda↗
PubMed1984

Transcription terminator involved in the expression of the int gene of phage lambda.

The phage lambda int gene is transcribed from two different promoters, pI and pL. Transcription from pI results in efficient synthesis of Int protein whereas transcription originating from pL results in poor int expression. The differential expression of Int from these two transcripts is dependent upon a site (sib) located distal to the int gene [Guarneros and Galindo, Virology 95 (1979) 119-126; Guarneros et al., Proc. Natl. Acad. Sci. USA 79 (1982) 238-242]. We have examined pI-promoted transcription in the region beyond the int coding sequence. The int mRNA extends to a site designated tI, which is located 277 nucleotides beyond int. Characterization of transcription at tI indicates that tI terminates with 75% efficiency in vitro, and that its efficiency is over 95% in vivo. The region between int and tI contains the regulatory signals needed for phage lamba integration and appears to be untranslated. The termination site overlaps with the sib control region that reduces Int synthesis from pL transcripts.

Bacteriophage lambda↗