Biomedical subjects

J B Kaplan

Publications and source records attributed to J B Kaplan.

At least 19 recordsLinked to original sources

PubMed2007

A survey of zoonotic pathogens carried by Norway rats in Baltimore, Maryland, USA.

Norway rats (Rattus norvegicus) carry several zoonotic pathogens and because rats and humans live in close proximity in urban environments, there exists potential for transmission. To identify zoonotic agents carried by rats in Baltimore, Maryland, USA, we live-trapped 201 rats during 2005-2006 and screened them for a panel of viruses, bacteria, and parasites. Antibodies against Seoul virus (57.7%), hepatitis E virus (HEV, 73.5%), Leptospira interrogans (65.3%), Bartonella elizabethae (34.1%), and Rickettsia typhi (7.0%) were detected in Norway rats. Endoparasites, including Calodium hepatica (87.9%) and Hymenolepis sp. (34.4%), and ectoparasites (13.9%, primarily Laelaps echidninus) also were present. The risk of human exposure to these pathogens is a significant public health concern. Because these pathogens cause non-specific and often self-limiting symptoms in humans, infection in human populations is probably underdiagnosed.

Animals↗

PubMed2007

Susceptibility of staphylococcal biofilms to enzymatic treatments depends on their chemical composition.

Bacterial infections are serious complications after orthopaedic implant surgery. Staphylococci, with Staphylococcus epidermidis as a leading species, are the prevalent and most important species involved in orthopaedic implant-related infections. The biofilm mode of growth of these bacteria on an implant surface protects the organisms from the host's immune system and from antibiotic therapy. Therapeutic agents that disintegrate the biofilm matrix would release planktonic cells into the environment and therefore allow antibiotics to eliminate the bacteria. An addition of a biofilm-degrading agent to a solution used for washing-draining procedures of infected orthopaedic implants would greatly improve the efficiency of the procedure and thus help to avoid the removal of the implant. We have previously shown that the extracellular staphylococcal matrix consists of a poly-N-acetylglucosamine (PNAG), extracellular teichoic acids (TAs) and protein components. In this study, we accessed the sensitivity of pre-formed biofilms of five clinical staphylococcal strains associated with orthopaedic prosthesis infections and with known compositions of the biofilm matrix to periodate, Pectinex Ultra SP, proteinase K, trypsin, pancreatin and dispersin B, an enzyme with a PNAG-hydrolysing activity. We also tested the effect of these agents on the purified carbohydrate components of staphylococcal biofilms, PNAG and TA. We found that the enzymatic detachment of staphylococcal biofilms depends on the nature of their constituents and varies between the clinical isolates. We suggest that a treatment with dispersin B followed by a protease (proteinase K or trypsin) could be capable to eradicate biofilms of a variety of staphylococcal strains on inert surfaces.

Bacterial Proteins↗

PubMed2005

Structural analysis of dispersin B, a biofilm-releasing glycoside hydrolase from the periodontopathogen Actinobacillus actinomycetemcomitans.

Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix that holds the bacteria together in a mass and firmly attaches the bacterial mass to the underlying surface. A major component of the extracellular polysaccharide matrix in several phylogenetically diverse bacteria is PGA, a linear polymer of N-acetylglucosamine residues in beta(1,6)-linkage. PGA is produced by the Gram-negative periodontopathogen Actinobacillus actinomycetemcomitans as well as by the Gram-positive device-associated pathogen Staphylococcus epidermidis. We recently reported that A.actinomycetemcomitans produces a soluble glycoside hydrolase named dispersin B, which degrades PGA. Here, we present the crystal structure of dispersin B at 2.0A in complex with a glycerol and an acetate ion at the active site. The enzyme crystallizes in the orthorhombic space group C222(1) with cell dimensions a=41.02A, b=86.13A, c=185.77A. The core of the enzyme consists a (beta/alpha)(8) barrel topology similar to other beta-hexosaminidases but significant differences exist in the arrangement of loops hovering in the vicinity of the active site. The location and interactions of the glycerol and acetate moieties in conjunction with the sequence analysis suggest that dispersin B cleaves beta(1,6)-linked N-acetylglucosamine polymer using a catalytic machinery similar to other family 20 hexosaminidases which cleave beta(1,4)-linked N-acetylglucosamine residues.

Acetylglucosamine↗

PubMed2002

Sequence diversity in the major fimbrial subunit gene (flp-1) of Actinobacillus actinomycetemcomitans.

Cells of the periodontal pathogen Actinobacillus actinomycetemcomitans exhibit tight adherence to surfaces such as glass, plastic and hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize teeth and other surfaces. Tight adherence is mediated by long fibrils of bundled pili (fimbriae) that form on the surface of the cell. The flp-1 gene encodes the major pilin protein component of A. actinomycetemcomitans fimbriae. In this study we compared flp-1 DNA sequences from 43 strains of A. actinomycetemcomitans isolated in Europe, Japan and the United States and identified seven distinct flp-1 allelic classes. DNA and predicted protein sequences were almost completely conserved within each flp-1 class but were highly divergent between classes. Most amino acid substitutions occurred in the C-terminus of the pilin protein, a region that has been shown to be important for the bundling and adhesive properties of the pili. flp-1 classes correlated with serotypes and 16S rRNA genotypes in most strains. At least five strains showed evidence of horizontal transfer of flp-1 between strains of different serotypes and 16S rRNA genotypes. Four of the seven flp-1 classes were present in geographically diverse isolates. Strains representing all seven flp-1 classes, but not a strain carrying a transposon insertion in flp-1, bound avidly to polystyrene in an in vitro adherence assay. Strains representing six of the seven flp-1 classes were isolated from localized juvenile periodontitis patients, suggesting that phylogenetically diverse strains carry pathogenic potential. Our findings provide a framework for future biochemical, immunological and genetic studies of A. actinomycetemcomitans fimbriae.

Actinobacillus Infections↗

PubMed2001

flp-1, the first representative of a new pilin gene subfamily, is required for non-specific adherence of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, a Gram-negative bacterium responsible for localized juvenile periodontitis and other infections such as endocarditis, produces long fibrils of bundled pili that are believed to mediate non-specific, tenacious adherence to surfaces. Previous investigations have implicated an abundant, small ( approximately 6.5 kDa), fibril-associated protein (Flp/Fap) as the primary fibril subunit. Here, we report studies on fibril structure and on the function and evolution of Flp. High-resolution electron microscopy of adherent clinical strain CU1000N revealed long bundles of 5- to 7-nm-diameter pili, whose subunits appear to be arranged in a helical array similar to that observed for type IV pili in other bacteria. Fibrils were found to be associated with the bacterial cell surface and smaller structures thought to be membrane vesicles. A modified version of the CU1000N Flp1 polypeptide with the T7-TAG epitope fused to its C-terminus was expressed in the wild-type strain, and the presence of the modified Flp1 in fibrils was confirmed by immunogold electron microscopy with monoclonal antibody to T7-TAG. To determine the importance of Flp1 in fibril formation and cell adherence, we used transposon IS903phikan to isolate insertion mutations in the flp-1 gene (formerly designated flp). Mutants with insertions early in flp-1 fail to produce fibrils and do not adhere to surfaces. Both fibril production and adherence were restored by cloned flp-1 in trans, thus providing the first evidence that flp-1 is required for fibril formation and tight, non-specific adherence. One mutant was found to have an insertion near the 3' end of flp-1 that results in the expression of a truncated and altered C-terminus of Flp1. This mutant produced short, unbundled pili, and its adherence to surfaces was significantly less than that of wild-type bacteria. These findings and related observations with the Flp1-T7-TAG protein indicate that the C-terminus of Flp1 is important for the bundling and adherence properties of pili. Extensive sequence comparisons and phylogenetic analysis of 61 predicted prepilin genes of bacteria revealed flp-1 to be a member of a novel and widespread subfamily of type IV prepilin genes. Thus, Flp pili are likely to be expressed by diverse bacterial species. Furthermore, we found that it is common for bacterial genomes to contain multiple alleles of flp-like genes, including the open reading frame (flp-2, previously designated orfA) immediately downstream of flp-1 in A. actinomycetemcomitans. The duplication and divergence of flp genes in bacteria may be important to the diversification of the colonization properties of these organisms.

Amino Acid Sequence↗

PubMed2001

Structural and genetic analyses of O polysaccharide from Actinobacillus actinomycetemcomitans serotype f.

The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP). A. actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from A. actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed of L-rhamnose and 2-acetamido-2-deoxy-D-galactose (molar ratio, 2:1) with the structure -->2)-alpha-L-Rhap-(1-3)-2-O-(beta-D-GalpNAc)-alpha-L-Rhap-(1-->* O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known A. actinomycetemcomitans serotypes. Strain CU1000 was mutagenized with transposon IS903phikan, and three mutants that were deficient in O-PS synthesis were isolated. All three transposon insertions mapped to a single 1-kb region on the chromosome. The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of A. actinomycetemcomitans serotype b, c, and e O-PS antigens. The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five known A. actinomycetemcomitans serotypes. These data indicate that strain CU1000 should be assigned to a new A. actinomycetemcomitans serotype, designated serotype f. A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored A. actinomycetemcomitans strains carrying the serotype f gene cluster. The finding of an A. actinomycetemcomitans serotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted.

Actinobacillus Infections↗

PubMed2001

Percutaneous core biopsy of nonpalpable breast lesions: utility and impact on cost of diagnosis.

Percutaneous image-guided core breast biopsy is faster, less invasive, less deforming, and less expensive than surgical biopsy for diagnosing nonpalpable breast lesions. Percutaneous biopsy may be performed using different imaging guidance modalities (e.g., stereotaxis or ultrasound) and different tissue acquisition devices (e.g., automated needles or vacuum-assisted biopsy probes). Stereotactic biopsy may be used for all lesion types (masses and calcifications). Stereotactic 14-gauge automated core biopsy spared a surgical procedure in 76-85% of cases, decreasing the cost of diagnosis by 40-56%. Annual national savings from use of stereotactic 14-gauge automated core biopsy rather than surgical biopsy for nonpalpable lesions would exceed 100 million dollars. Stereotactic 11-gauge vacuum-assisted biopsy yields significant improvement in diagnosis of calcifications and may be used in lesions that are not amenable to stereotactic 14-gauge automated core biopsy. Stereotactic 11-gauge vacuum-assisted biopsy spared a surgical procedure in 76% cases, decreasing cost of diagnosis by 20%. Use of stereotactic 11-gauge vacuum-assisted biopsy for calcifications and for nonpalpable masses not amenable to stereotactic 14-gauge automated core biopsy would yield annual national savings exceeding 50 million dollars. Ultrasound-guided biopsy, used primarily for masses, has several advantages including speed, comfort, lack of radiation exposure, and real-time needle visualization. Ultrasound-guided core biopsy spared a surgical procedure in 85% cases, decreasing the cost of diagnosis by 56%. Although both ultrasound-guided core biopsy and stereotactic biopsy are less expensive than surgery, cost savings are greater for ultrasound-guided core biopsy. An annual national savings over $50 million could be realized with the use of ultrasound-guided core biopsy for nonpalpable masses. Future work should include evaluating the emerging new technologies for percutaneous breast biopsy and optimizating the choice of biopsy method for different clinical scenarios. Society may benefit from cost reduction as women benefit from a faster, less invasive, and less deforming approach to diagnosis.

Journal Article↗

PubMed1998

Codon usage in Actinobacillus actinomycetemcomitans.

The codon usage patterns of 21 genes encompassing 5800 codons from Actinobacillus actinomycetemcomitans were analyzed. A. actinomycetemcomitans genes could be divided into two groups based on their function and G + C content. One group included those genes encoding basic cellular functions. This group displayed an average G + C content of 48%. A second group comprised genes encoding the leukotoxin determinant, an insertion sequence and a plasmid. This group displayed an average G + C content of 36%. These findings suggest that portions of the A. actinomycetemcomitans genome may have been acquired by horizontal gene transfer from one or more distantly related species. We present a table of A. actinomycetemcomitans codon usage. These data may be used to establish standards for computer programs that predict A. actinomycetemcomitans protein coding regions and may be useful in designing degenerate oligonucleotide probes.

Adolescent↗

PubMed1997

Characterization of a soluble vascular endothelial growth factor receptor-immunoglobulin chimera.

To investigate the interaction between vascular endothelial growth factor (VEGF) and its receptor, we have constructed a chimeric protein consisting of the extracellular ligand-binding domain of the human VEGF receptor subtype KDR fused to a human IgG1 Fc domain (KDR-Fc). KDR-Fc was expressed in human 293 kidney epithelial cells as a 300-kDa secreted, dimeric glycoprotein that bound 125I-VEGF165 with high affinity (Kd = 150 pM). Unlike the full length cellular receptor, KDR-Fc did not require heparin for 125I-VEGF165 binding, although heparin did stimulate 125I-VEGF165 binding approximately 50 to 100%. Similar results were observed for KDR-Fc expressed in yeast cells. Since yeast do not synthesize heparan sulfate proteoglycans, we conclude that cellular heparan sulfates do not account for the lack of a heparin requirement for 125I-VEGF165 binding to KDR-Fc. The polycationic protein protamine, which inhibits (IC50 = 1 microgram/ml) 125I-VEGF165 binding to bovine aortic endothelial cells and other KDR-expressing cells by blocking heparin interactions, had no effect on the heparin independent component of 125I-VEGF165 binding to KDR-Fc. Protamine does inhibit (IC50 = 1 microgram/ml) the heparin dependent component of 125I-VEGF165 binding to KDR-Fc. KDR-Fc bound VEGF121 with the same affinity as VEGF165. Heparin had no effect on 125I-VEGF121 binding to KDR-Fc, indicating that heparin interaction with the 44 amino acids contained in VEGF165 but not VEGF121 allow for maximal VEGF165 binding. Deletion analysis of KDR-Fc demonstrated that the determinants required for high affinity VEGF binding are located in the three aminoterminal Ig-domains of the protein. Heparin had no effect on 125I-VEGF165 binding to the three Ig-domain receptor, suggesting that there are heparin binding determinants located in KDR Ig-domains 4 to 7.

Amino Acid Sequence↗

PubMed1994

Biological activity of human N-ras and K-ras genes containing the Asn17 dominant negative mutation.

Substitution of asparagine for serine at position 17 of human H-ras results in an impaired GTP-binding activity, causing the mutant Ras protein to be locked in a constitutively inactive GDP-bound state. Expression of this mutant in NIH 3T3 cells inhibits cell proliferation by blocking endogenous ras function. Plasmids that encode the analogous dominant negative mutation at position 17 in human N- and K-ras were constructed. These mutant ras genes, driven by a heavy metal-inducible sheep metallothionein promoter, were introduced by transfection into a variety of animal cell lines. All three mutant ras genes displayed an inhibitory phenotype when expressed in NIH 3T3 cells. This inhibition could be overcome by cotransfection with either activated H-ras or v-raf. These data indicate that the three human Ras proteins probably act through the same signal transduction pathway in NIH 3T3 cells and suggest that these mutations may confer similar phenotypes to other GTP/GDP-binding proteins.

3T3 Cells↗

PubMed1993

Influence of season on seminal characteristics, testis size and serum testosterone in the western spotted skunk (Spilogale gracilis).

The western spotted skunk is a seasonally breeding mammal: most copulations occur in late September and early October. This study was performed to characterize the seasonal changes in concentrations of testosterone and in ejaculate quality. Captive males (n = 22) were maintained on a natural photoperiod for 15 months. Semen samples were collected by electroejaculation; testis size was measured; and blood samples were collected. Of 110 electroejaculation attempts, 104 (95%) resulted in successful fluid collection and 101 (97%) samples contained spermatozoa. Significant increases (P < 0.05) in serum testosterone concentration, testis size and ejaculate volume were observed from August to November. Mean concentration of testosterone in serum ranged from 0.15 +/- 0.05 ng ml-1 in mid-January to 6.42 +/- 1.79 ng ml-1 in early October. Mean testis size ranged from 1.22 +/- 0.25 cm2 in February to 2.68 +/- 0.08 cm2 in October. Mean ejaculate volume ranged from 11 +/- 3 microliters in March to 129 +/- 22 microliters in October. Seasonal changes in the number of spermatozoa per ejaculate or motility of spermatozoa were not observed. Mean number of spermatozoa per ejaculate was 8.14 +/- 0.85 x 10(6) spermatozoa (n = 97); motility was 56 +/- 2.4% (n = 93); semen pH was 7.76 +/- 0.20 (n = 6); osmolarity was 394 +/- 13 mmol kg-1 (n = 10); and 70.3 +/- 1.5% of the spermatozoa were morphologically normal (n = 47).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Phylogenetic classification of human papillomaviruses: correlation with clinical manifestations.

Human papillomaviruses (HPVs) are a heterogeneous group of small dsDNA viruses which cause a variety of proliferative epithelial lesions at specific anatomical sites. Although more than 65 different virus types have been cloned and characterized, no uniform classification system exists. In order to classify HPV DNA types, phylogenetic trees were constructed based on nucleotide sequence alignments using parsimony and distance matrix algorithms. The resulting phylogenetic trees provide a classification of the HPVs into specific groups encompassing the known tissue tropism and oncogenic potential of each HPV type. The implications of a phylogenetic taxonomy on the diagnostic detection of HPVs and the concept of different HPV species are discussed.

Algorithms↗

PubMed1991

Prolactin levels in the western spotted skunk: changes during pre- and periimplantation and effects of melatonin and lesions to the anterior hypothalamus.

Prolactin (PRL) is the primary pituitary hormone responsible for initiating increased function of the corpus luteum and blastocyst implantation in the western spotted skunk. Therefore, we have designed experiments to validate a PRL RIA, characterize the preimplantation profile in PRL secretion, and determine the effects of exogenous melatonin and lesions to the anterior hypothalamic area (AHA) on PRL secretion in the skunk. These objectives were investigated with a heterologous RIA using canine PRL standards and antiserum. Displacement curves of skunk pituitary extract and serum were parallel to the canine PRL standard curve. Growth hormone-releasing hormone injection did not cause a significant change in plasma PRL levels as detected by the assay (p = 0.74). Injection of pimozide increased and bromocriptine decreased plasma PRL levels (p less than 0.05). A seasonal trend in plasma PRL levels was observed during the preimplantation period, with mean concentrations ranging from 5 ng/ml during the period of short day length in January to 17.1 ng/ml during the long day photoperiod in early May. The average date of blastocyst implantation in this study was 2 May (n = 16). Silastic capsules containing melatonin (n = 5) significantly delayed both the seasonal rise in plasma PRL levels and the time of implantation (p less than 0.05) compared to controls with empty capsules (n = 4). Lesions to the AHA (AHAx, n = 5) eliminated these effects of melatonin on both the rise in PRL and time of implantation. PRL levels were highly correlated with progesterone levels (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Post-translational processing of purified human K-ras in Xenopus oocytes.

Membrane localization of ras p21 involves a complex series of post-translational processing events, including S-farnesylation of Cys-186, removal of three carboxyl-terminal amino acid residues, and methylation of the carboxyl-terminal farnesylcysteine residue. Palmitoylation of cysteine residues within the hypervariable region (amino acids 165-185) is also required for membrane localization of mammalian H-, N-, and K-ras(A). For K-ras(B), which contains no cysteine residues within the hypervariable region, a polybasic domain substitutes for palmitoylation as a second signal for plasma membrane targeting. In order to investigate the localization of K-ras(B) to the plasma membrane, we purified wild-type and mutant human K-ras(B) proteins from strains of E. coli harboring bacterial expression plasmids and injected them into Xenopus laevis oocytes. Our results show that wild-type and activated K-ras(B) proteins can be post-translationally modified and can induce meiotic maturation in Xenopus oocytes. A mutation at Cys-186 (Cys to Gly) abolished the ability of activated K-ras(B) to induce meiosis. Deprivation of isoprenyl precursors by the addition of lovastatin, a drug that blocks the synthesis of mevalonate, also abolished the ability of activated K-ras(B) to induce meiosis, although this inhibition could be overcome by the addition of exogenous mevalonate. Lovastatin did not block meiotic maturation induced by microinjection of purified mos protein, a component of the cytostatic factor that arrests Xenopus oocytes at the first meiotic prophase. These results indicate that post-translational isoprenylation of K-ras(B) is essential for plasma membrane targeting and induction of meiotic maturation in Xenopus oocytes and that further isoprenyl modification of proteins downstream from mos signal transduction is not essential for this process.

Animals↗

PubMed1990

Platelet-activating factor or a platelet-activating factor antagonist decreases tumor necrosis factor-alpha in the plasma of mice treated with endotoxin.

When L-platelet-activating factor (PAF) or alprazolam (a PAF antagonist) was administered to lipopolysaccharide (LPS)-treated mice, the level of plasma tumor necrosis factor (TNF alpha) determined by either ELISA or a cytotoxic assay using WEHI cells was significantly lowered. The inactive stereoisomer, D-PAF, was not effective in lowering plasma TNF alpha levels in LPS-treated mice. The decrease in plasma TNF alpha induced by L-PAF or alprazolam was partly reversed by indomethacin. Despite a decrease in plasma TNF alpha, L-PAF or alprazolam caused an increase in the amount of TNF alpha mRNA present in the kidneys and the livers of LPS-treated mice, suggesting that a posttranscriptional event leading to the synthesis or release of TNF alpha was inhibited by these agents.

Alprazolam↗

PubMed1990

An evolutionary comparison of Acinetobacter calcoaceticus trpF with trpF genes of several organisms.

The deduced amino acid sequence of Acinetobacter calcoaceticus N-(5'-phosphoribosyl) anthranilate isomerase (PRAI), which is coded by trpF, was compared with TrpF of Caulobacter crescentus, Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Neurospora crassa, and Aspergillus nidulans. Sixty percent of identical or similar amino acids were located in alpha/beta TIM (triose-phosphate isomerase) barrels and in residues important in substrate binding and catalysis. In addition, the analysis of trpF genes presented here supports a model by which fusion between separate trpC and trpF genes arose in some cases by in-frame deletions.

Acinetobacter↗

PubMed1989

Temporal regulation and overlap organization of two Caulobacter flagellar genes.

The biogenesis of the bacterial flagellum and chemotaxis apparatus in both Escherichia coli and Caulobacter crescentus requires the ordered expression of over 40 genes whose expression is controlled by a trans-acting regulatory hierarchy. In C. crescentus, additional control mechanisms ensure that the transcription of these genes is initiated at the correct time in the cell cycle. We demonstrate here that two flagellar genes, flaE and flaY, whose products function in trans to modulate the level of transcription of other flagellar genes, are themselves temporally controlled. DNA sequence analysis of the 3413 base-pairs encompassing the flaE and flaY coding sequences and the 5' regulatory region showed that flaE encodes a protein of 16,000 Mr and flaY a protein of 17,000 Mr. Evidence that flaE and flaY are transcribed as a polycistronic message includes (1) the polar effect of Tn5 insertions; (2) deletion analysis showing that the flaE promoter is essential for complementation of both flaE and flaY alleles; and (3) nuclease S1 assays showing protection of a transcript spanning both genes. The transcript start site in front of flaE was determined and the -10 region conforms to the E. coli sigma 28 promoter consensus sequence. Nuclease S1 analysis also revealed a protected fragment whose size was consistent with a transcript initiating in vivo at a consensus "nif" promoter sequence in front of the flaY gene. The entire promoter region and an upstream consensus sequence that might be a regulatory element for the flaY gene lies within the carboxyl-terminal coding sequence of the flaE gene.

Amino Acid Sequence↗

PubMed1987

Molecular analysis of integrated human papillomavirus 16 sequences in the cervical cancer cell line SiHa.

Human papillomavirus (HPV) 16 is frequently found integrated into cervical cancer cell genomes and these integrations are thought to play a role in tumorigenesis. To investigate the mechanisms of HPV integration and its effect on transcription and chromosomal sequence organization, we have cloned and analyzed the HPV16 integration from the cervical cancer cell line SiHa. Restriction analyses and Southern blotting indicated that approximately 95% of an HPV16 genome was integrated without gross rearrangement. Sequence analysis of the cellular-viral DNA junctions revealed that integration had occurred within the E2 and E4 ORFs where 251 bp of viral sequence was deleted. One viral terminus occurred within sequences of an Alu repeat and a 4-bp homology was present at the site of recombination. Using unique cellular flanking DNA probes, a 4.8-kb deletion of cellular sequences was detected at the site of viral integration. The chromosomal location of the viral integration and cellular deletion were mapped to chromosome 13 using a rodent X human somatic cell hybrid panel. Northern blot analysis using viral subgenomic and 3' cellular probes revealed transcription from the 3' portion of integrated HPV16 (E6, E7, E1) and flanking cellular sequences. The observation of viral-cell transcripts and chromosomal deletions associated with HPV integration may indicate that such events are part of a multistep mechanism leading to the development of cervical cancer.

Base Sequence↗