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PubMed1997

Allelic polymorphism of two multifunctional regions in the central human MHC: tenascin X, XB-S and YB, and their duplicated fragments XA and YA.

Two highly polymorphic sequences have been discovered in the complement C4 region of the human major histocompatibility complex (MHC). They are part of a duplicated unit of overlapping genes, transcribed in opposite directions and containing the sequences of tenascin X (XB), XB-S, XA, YB and YA. Fragments of 1014 bp and 894 bp were co-amplified by polymerase chain reaction (PCR) and digested with two different restriction enzymes. This preliminary study provides evidence for more than five different alleles each of XA (YA) and XB (XB-S, YB), and at least 11 XA-XB haplotypes. Their association with extended HLA-B-DR haplotypes, C4 complotypes, and C4 region restriction fragment length polymorphisms (RFLP) is discussed. X gene polymorphisms are a complement region marker system that should be uniquely suited for PCR-based typing methods. They could become a useful addition to HLA class I and class II markers in the mapping of candidate genes for MHC-associated diseases, including the X and Y genes themselves.

Alleles↗
PubMed1991

Three in-frame N-terminally different proteins are produced from the repressor locus of the Streptomyces bacteriophage phi C31.

The sequence of the repressor locus, c, of the Streptomyces temperate phage, phi C31, was shown previously to contain an open reading frame encoding a 74 kDa protein. Further analysis of the transcriptional and translational products of the c gene shows a more complex pattern of expression. A nest of three in-frame N-terminally different, C-terminally identical proteins of 74, 54 and 42 kDa were found to be expressed from a corresponding nest of transcripts. The repressor proteins were produced in Escherichia coli and the 42 kDa protein was purified, verified by N-terminal sequencing, and used to raise antibody. The antibody cross-reacted in Western blots with the 74, 54 and 42 kDa proteins expressed in E. coli and Streptomyces lividans and from Streptomyces coelicolor phi C31 lysogens. Analysis of transcription of the c gene by S1 mapping and primer extension showed that the nest of transcripts encoding the repressor protein were induced after heat treatment of the cts locus (Sinclair and Bibb, 1989; this paper). Correspondingly, all three of the repressor proteins were induced. In addition to a promoter, cp1, which lies upstream of the 74 kDa open reading frame, the c locus contained at least one internal promoter, cp2, which transcribes DNA encoding the 54 and 42 kDa proteins. Transcripts initiating from cp3 were observed in RNA preparations from S. lividans containing the c gene deleted for cp1 and cp2, but gene fusions using DNA which should contain any putative promoting activity from this region transcriptionally fused to the xylE gene showed very low levels of expression of catechol 2,3 dioxygenase in S. lividans. The 74 kDa protein was not necessary for super-infection immunity. Data described here and current knowledge of the nature of other 'dual start' genes suggest a model for the regulation of lysis versus lysogeny in phi C31.

Bacteriophages↗
PubMed1992

Identification of replication and stability functions in the complete nucleotide sequence of plasmid pUH24 from the cyanobacterium Synechococcus sp. PCC 7942.

The complete nucleotide sequence is presented for pUH24, the small plasmid of Synechococcus sp. PCC 7942. pUH24 consists of 7835bp and has a G + C content of 59%. The distribution of translation start and stop codons in the sequence allows 36 open reading frames that potentially encode polypeptides of 50 or more amino acids. We postulate that eight of these open reading frames are actual coding sequences. A region has been identified, by experiment, that contains two functions, designated pmaA and pmaB, involved in the segregational stability of the plasmid. The minimal region of pUH24 fully capable of supporting autonomous replication consists of a 3.6kb DNA fragment, which is almost entirely occupied by two overlapping genes most likely coding for essential replication proteins (repA and repB).

Amino Acid Sequence↗
PubMed2005

A genetic analysis of ambulatory cardiorespiratory coupling.

This study assessed the heritability of ambulatory heart period, respiratory sinus arrhythmia (RSA), and respiration rate and tested the hypothesis that the well-established correlation between these variables is determined by common genetic factors. In 780 healthy twins and siblings, 24-h ambulatory recordings of ECG and thorax impedance were made. Genetic analyses showed considerable heritability for heart period (37%-48%), RSA (40%-55%), and respiration rate (27%-81%) at all daily periods. Significant genetic correlations were found throughout. Common genes explained large portions of the covariance between heart period and RSA and between respiration rate and RSA. During the afternoon and night, the covariance between respiration rate and RSA was completely determined by common genes. This overlap in genes can be exploited to increase the power of linkage studies to detect genetic variation influencing cardiovascular disease risk.

Adult↗
PubMed1989

Identification of the cystic fibrosis gene: chromosome walking and jumping.

An understanding of the basic defect in the inherited disorder cystic fibrosis requires cloning of the cystic fibrosis gene and definition of its protein product. In the absence of direct functional information, chromosomal map position is a guide for locating the gene. Chromosome walking and jumping and complementary DNA hybridization were used to isolate DNA sequences, encompassing more than 500,000 base pairs, from the cystic fibrosis region on the long arm of human chromosome 7. Several transcribed sequences and conserved segments were identified in this cloned region. One of these corresponds to the cystic fibrosis gene and spans approximately 250,000 base pairs of genomic DNA.

Animals↗
PubMed1989

Mutations in the hemolytic-phospholipase C operon result in decreased virulence of Pseudomonas aeruginosa PAO1 grown under phosphate-limiting conditions.

The phospholipase C (PLC) operon of Pseudomonas aeruginosa consists of plcS, which encodes a heat-labile secreted hemolysin, and two in-phase, overlapping genes, plcR1 and plcR2, which may encode Pi-regulatory genes. A 2.8-kilobase-pair deletion mutation in this operon was constructed, and a tetracycline resistance (Tcr) cartridge replaced the deleted sequences. A deletion mutant of strain PAO1 was obtained through recombination between the flanking regions of the mutated cloned PLC operon and the homologous chromosomal regions. The deletion of the chromosomal PLC operon and its replacement by the Tcr cartridge was confirmed by Southern hybridization. The deletion strain, PLC SR, is nonhemolytic. However, it retains PLC activity when measured on a synthetic substrate. A second mutant strain, PLC R, contains a deletion in the plcR genes. This mutant is more hemolytic and produces more enzymatic activity than PAO1. The virulence of both of these mutants was compared with that of PAO1 in the mouse burn model of infection. When mice were infected with cultures grown in a high-Pi medium, there was a 10-fold increase in the 50% lethal dose of the mutants compared with PAO1. In contrast, when the inoculum originated from low-Pi cultures, there was a 200- to 10,000-fold increase in the 50% lethal dose of the mutants over PAO1.

Animals↗
PubMed1995

Interactions of the origin of replication (oriV) and initiation proteins (TrfA) of plasmid RK2 with submembrane domains of Escherichia coli.

It has been possible to locate a submembrane domain representing less than 10% of the total membrane that appears to be responsible for sequestering some essential components required for plasmid RK2 DNA replication. This subfraction, whose cellular location in the membrane prior to extraction is still unknown, is derived from the inner membrane fraction, since it possesses enzyme marker activity (NADH oxidase) exclusively associated with the inner membrane. The subfraction was detected by a modification of the methods of Ishidate et al. (K. Ishidate, E. S. Kreeger, J. Zrike, S. Deb, B. Glauner, T. MacAlister, and L. I. Rothfield, J. Biol. Chem. 261:428-443, 1986) in which low pressure in a French pressure cell and lysozyme were used to preserve the supercoil plasmid DNA template during cell disruption. This was followed by successive cycles of sucrose gradient sedimentation and flotation density gradient centrifugation to reveal a number of subfractions, including the one of interest. The characteristics of plasmid interaction with the subfraction include the presence of supercoil DNA after extraction, the binding of the origin of plasmid replication (oriV) in vitro, and the association of the two plasmid-encoded initiation (TrfA) proteins (encoded by overlapping genes). However, another peak, the outer membrane fraction, also binds oriV in vitro, contains plasmid DNA in vivo, and associates with the TrfA initiation proteins. Nevertheless, it contains much less of the initiation proteins, and the specific activity of binding oriV is also much reduced compared with the other subfraction. There is a strong correlation between the association of the TrfA initiation proteins with a particular membrane fraction and the binding of oriV in vitro or plasmid DNA in vivo. Since the proteins are known to bind to repeated sequences in oriV (S. Perri, D. R. Helinski, and A. Toukdarian, J. Biol. Chem. 266:12536-1254, 1991; M. Pinkney, R. Diaz, E. Lanka, and C. M. Thomas, J. Mol. Biol. 203: 927-938, 1988), it appears that the initiation proteins themselves could be responsible, at least in part, for the association of plasmid DNA to the membrane.

Bacterial Proteins↗
PubMed1998

A family of stability determinants in pathogenic bacteria.

A novel segregational stability system was identified on plasmid R485, which originates from Morganella morganii. The system is composed of two overlapping genes, stbD and stbE, which potentially encode proteins of 83 and 93 amino acids, respectively. Homologs of the stbDE genes were identified on the enterotoxigenic plasmid P307 from Escherichia coli and on the chromosomes of Vibrio cholerae and Haemophilus influenzae biogroup aegyptius. The former two homologs also promote plasmid stability in E. coli. Furthermore, the stbDE genes share homology with components of the relBEF operon and with the dnaT gene of E. coli. The organization of the stbDE cassette is reminiscent of toxin-antitoxin stability cassettes.

Amino Acid Sequence↗
PubMed1998

Campylobacter fetus surface layer proteins are transported by a type I secretion system.

The virulence of Campylobacter fetus, a bacterial pathogen of ungulates and humans, is mediated in part by the presence of a paracrystalline surface layer (S-layer) that confers serum resistance. The subunits of the S-layer are S-layer proteins (SLPs) that are secreted in the absence of an N-terminal signal sequence and attach to either type A or B C. fetus lipopolysaccharide in a serospecific manner. Antigenic variation of multiple SLPs (encoded by sapA homologs) of type A strain 23D occurs by inversion of a promoter-containing DNA element flanked by two sapA homologs. Cloning and sequencing of the entire 6.2-kb invertible region from C. fetus 23D revealed a probable 5.6-kb operon of four overlapping genes (sapCDEF, with sizes of 1,035, 1,752, 1,284, and 1,302 bp, respectively) transcribed in the opposite direction from sapA. The four genes also were present in the invertible region of type B strain 84-107 and were virtually identical to their counterparts in the type A strain. Although SapC had no database homologies, SapD, SapE, and SapF had predicted amino acid homologies with type I protein secretion systems (typified by Escherichia coli HlyBD/TolC or Erwinia chrysanthemi PrtDEF) that utilize C-terminal secretion signals to mediate the secretion of hemolysins, leukotoxins, or proteases from other bacterial species. Analysis of the C termini of four C. fetus SLPs revealed conserved structures that are potential secretion signals. A C. fetus sapD mutant neither produced nor secreted SLPs. E. coli expressing C. fetus sapA and sapCDEF secreted SapA, indicating that the sapCDEF genes are sufficient for SLP secretion. C. fetus SLPs therefore are transported to the cell surface by a type I secretion system.

Amino Acid Sequence↗
PubMed2001

Transcriptional analysis of the tet(P) operon from Clostridium perfringens.

The Clostridium perfringens tetracycline resistance determinant from the 47-kb conjugative R-plasmid pCW3 is unique in that it consists of two overlapping genes, tetA(P) and tetB(P), which mediate resistance by different mechanisms. Detailed transcriptional analysis has shown that the inducible tetA(P) and tetB(P) genes comprise an operon that is transcribed from a single promoter, P3, located 529 bp upstream of the tetA(P) start codon. Deletion of P3 or alteration of the spacing between the -35 and -10 regions significantly reduced the level of transcription in a reporter construct. Induction was shown to be mediated at the level of transcription. Unexpectedly, a factor-independent terminator, T1, was detected downstream of P3 but before the start of the tetA(P) gene. Deletion or mutation of this terminator led to increased read-through transcription in the reporter construct. It is postulated that the T1 terminator is an intrinsic control element of the tet(P) operon and that it acts to prevent the overexpression of the TetA(P) transmembrane protein, even in the presence of tetracycline.

Antiporters↗
PubMed2002

Failure of commercial ligase chain reaction to detect Mycobacterium tuberculosis DNA in sputum samples from a patient with smear-positive pulmonary tuberculosis due to a deletion of the target region.

We report on a strain of Mycobacterium tuberculosis with a deletion in the protein antigen B gene overlapping the probe binding sites for the Abbott Diagnostics LCx M. tuberculosis (LCx-MTB) probe assay. A false-negative result with the LCx-MTB assay delayed a laboratory diagnosis of tuberculosis.

Antigens, Bacterial↗
PubMed2006

High-throughput, library-based selection of a murine leukemia virus variant to infect nondividing cells.

Gammaretroviruses, such as murine leukemia virus (MLV), are functionally distinguished from lentiviruses, such as human immunodeficiency virus, by their inability to infect nondividing cells. Attempts to engineer this property into MLV have been hindered by an incomplete understanding of early events in the viral life cycle. We utilized a transposon-based method to generate saturated peptide insertion libraries of MLV gag-pol variants with nuclear localization signals randomly incorporated throughout these overlapping genes. High-throughput selection of the libraries via iterative retroviral infection of nondividing cells led to the identification of a novel variant that successfully transduced growth-arrested cells. Vector packaging by cotransfection of the gag-pol.NLS variant with wild-type gag-pol produced high-titer virions capable of infecting neurons in vitro and in vivo. The capacity of mutant virions to transduce nondividing cells could help to elucidate incompletely understood mechanisms of the viral life cycle and greatly broaden the gene therapy applications of retroviral vectors. Furthermore, the ability to engineer key intracellular viral infection steps has potential implications for the understanding, design, and control of other post-entry events. Finally, this method of library generation and selection for a desired phenotype directly in a mammalian system can be readily expanded to address other challenges in protein engineering.

Active Transport, Cell Nucleus↗
PubMed1981

Biochemical, biophysical, and biological properties of densonucleosis virus (parvovirus). III. common sequences of structural proteins.

Densonucleosis virus cannot code for its four structural proteins if each of them has a unique sequence. The objective of the present investigation, therefore, was to establish whether: (i) the viral genome contains overlapping genes; (ii) the virus incorporates host proteins; or (iii) one of the structural proteins is a dimer. Two independent methods were employed for this purpose. First, the viral proteins, solubilized in sodium dodecyl sulfate, were purified after dansylation and were analyzed by peptide mapping, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Second, an enzyme-linked immunosorbent assay was developed for a comparative analysis of the viral proteins solubilized by sodium dodecyl sulfate. It was demonstrated with both techniques that densonucleosis virus has four unique structural proteins, all with extensive sequence homologies. Moreover, all structural proteins contained intraprotein, but no interprotein, disulfide linkages. These results indicated similarities between densonucleosis virus and representatives of the two other genera of the Parvoviridae.

Amino Acid Sequence↗
PubMed1987

In vivo transcription of bacteriophage phi 29 DNA: transcription termination.

The main early and late transcription termination sites in vivo in bacteriophage phi 29 DNA were determined by nuclease S1 mapping. Transcription of the phi 29 early genes located at the left end of the viral genome terminated at the very end of the DNA molecule and within the HindIII G fragment of the viral DNA. Transcription termination of the early genes located at the right end of the genome and that of the late viral genes overlapped in a specific region of the phi 29 DNA within the EcoRI D fragment. Stem-loop structures followed by uridine-rich tails could be derived close to the 3' ends of early and late mRNAs, suggesting Rho-independent transcription termination in phi 29 DNA.

Bacteriophages↗
PubMed1991

An early gene maps within and is 3' coterminal with the immediate-early gene of equine herpesvirus 1.

The immediate-early (IE) gene (IR1 gene) of equine herpesvirus 1 (EHV-1) encodes a single, spliced 6.0-kb mRNA during cytolytic infection. However, under early (in the presence of phosphonoacetic acid) and late (8 h postinfection; no metabolic inhibitors) conditions, in addition to the 6.0-kb IE mRNA, a 4.4-kb early (E) mRNA is transcribed from the IE gene region beginning at approximately 4 h postinfection. To map and characterize the 4.4-kb E mRNA and the protein product of this early gene (IR2 gene), Northern (RNA) blot hybridization, S1 nuclease, primer extension, and in vitro transcription and translation analyses were used. The data from RNA mapping analyses revealed that the 4.4-kb E IR2 mRNA (i) maps at nucleotides 4481 to 635 within each of the inverted repeats of the short region and thus is encoded by sequences that lie entirely within the IE gene, (ii) is transcribed in the same direction as the IE mRNA, initiating at nucleotide 4481, which lies 25 bp downstream of a putative TATA-like sequence and 1,548 bp downstream of the transcription initiation site of the IE mRNA, and (iii) is 3' coterminal with the IE mRNA which terminates at nucleotide 635 of the inverted repeats. The IR2 open reading frame was inserted into the transcription expression vector pGEM-3Z, and the RNA transcribed from this construct (pGEM44) was shown to be a 4.2-kb transcript that contained all IR2 sequences. In vitro translation of the 4.2-kb RNA yielded a major protein of approximately 130 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. This protein corresponds to the predicted IR2 product of 1,165 amino acids that would be in frame with the major IE polypeptide (IE1 = 200 kDa; 1,487 amino acids) and thus would be a 5'-truncated form of the IE1 polypeptide. The presence and potential role of the IR2 gene embedded within the IR1 gene increase the complexity of the regulation of the IE gene region during various stages of a productive infection.

Base Sequence↗
PubMed1996

The spontaneous reactivation function of the herpes simplex virus type 1 LAT gene resides completely within the first 1.5 kilobases of the 8.3-kilobase primary transcript.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation of HSV-1 from latency. We report here that although the LAT gene is 8.3 kb in length, the first 1.5 kb of the LAT gene alone is sufficient for wild-type levels of spontaneous reactivation. We began with a LAT deletion mutant of HSV-1 strain McKrae in which the LAT promoter and the first 1.6 kb of the 5' end of the LAT gene had been deleted from both copies of LAT (one in each viral long repeat). As we previously reported, this mutant (dLAT2903) was significantly impaired for spontaneous reactivation (G. C. Perng, E. C. Dunkel, P. A. Geary, S. M. Slanina, H. Ghiasi, R. Kaiwar, A. B. Nesburn, and S. L. Wechsler, J. Virol. 68:8045-8055, 1994). We then inserted the LAT promoter and the first 1.5 kb of the LAT gene into a location in the unique long region of dLAT2903 far removed from the normal location of LAT in the long repeats. This resulted in a virus (LAT15a) whose capacity for transcribing LAT RNA was limited to the first 1.5 kb of the 8.3-kb LAT primary transcript. Rabbits were ocularly infected with this mutant, and spontaneous reactivation was measured in comparison to those of the original LAT-negative mutant and its marker-rescued (wild-type) virus, dLAT2903R. LAT15a had an in vivo spontaneous reactivation rate of 12%, compared with a rate of 11% for the marker-rescued virus and 0% for the LAT-negative virus. Southern analysis confirmed that the spontaneously reactivated LAT15a virus retained the original deletions in both copies of LAT and the 1.5-kb LAT insertion in the unique long region. Thus, insertion of the first 1.5 kb of LAT (and its promoter) at a site distant from the normal LAT location appeared to completely restore in vivo spontaneous reactivation to wild-type levels, despite the remaining inability of the original LAT genes to transcribe any LAT RNA. The function of LAT involved in efficient spontaneous reactivation therefore appeared to map completely within the first 1.5 kb of the LAT gene.

Animals↗
PubMed1999

Comparison of genetic variability at multiple loci across the genomes of the major subtypes of Kaposi's sarcoma-associated herpesvirus reveals evidence for recombination and for two distinct types of open reading frame K15 alleles at the right-hand end.

Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV8) DNA is found consistently in nearly all classical, endemic, transplant, and AIDS-associated KS lesions, as well as in several AIDS-associated lymphomas. We have previously sequenced the genes for the highly variable open reading frame K1 (ORF-K1) protein from more than 60 different HHV8 samples and demonstrated that they display up to 30% amino acid variability and cluster into four very distinct evolutionary subgroups (the A, B, C, and D subtypes) that correlate with the major migrationary diasporas of modern humans. Here we have extended this type of analysis to six other loci across the HHV8 genome to further evaluate overall genotype patterns and the potential for chimeric genomes. Comparison of the relatively conserved ORF26, T0.7/K12, and ORF75 gene regions at map positions 0. 35, 0.85, and 0.96 revealed typical ORF-K1-linked subtype patterns, except that between 20 and 30% of the genomes analyzed proved to be either intertypic or intratypic mosaics. In addition, a 2,500-bp region found at the extreme right-hand side of the unique segment in 45 HHV8 genomes proved to be highly diverged from the 3,500-bp sequence found at this position in the other 18 HHV8 genomes examined. Furthermore, these previously uncharacterized "orphan" region sequences proved to encompass multiexon latent-state mRNAs encoding two highly diverged alleles of the novel ORF-K15 protein. The predominant (P) and minor (M) forms of HHV8 ORF-K15 are structurally related integral membrane proteins that have only 33% overall amino acid identity to one another but retain conserved likely tyrosine kinase signaling motifs and may be distant evolutionary relatives of the LMP2 latency protein of Epstein-Barr virus. The M allele of ORF-K15 is also physically linked to a distinctive M subtype of the adjacent ORF75 gene locus, and in some cases, this linkage extends as far back as the T0.7 locus also. Overall, the results suggest that an original recombination event with a related primate virus from an unknown source introduced exogenous right-hand side ORF-K15(M) sequences into an ancient M form of HHV8, followed by eventual acquisition into the subtype C lineage of the modern P-form of the HHV8 genome and subsequent additional, more recent transfers by homologous recombination events into several subtype A and B lineages as well.

Alleles↗
PubMed2005

Predicting coding potential from genome sequence: application to betaherpesviruses infecting rats and mice.

Prediction of protein-coding regions and other features of primary DNA sequence have greatly contributed to experimental biology. Significant challenges remain in genome annotation methods, including the identification of small or overlapping genes and the assessment of mRNA splicing or unconventional translation signals in expression. We have employed a combined analysis of compositional biases and conservation together with frame-specific G+C representation to reevaluate and annotate the genome sequences of mouse and rat cytomegaloviruses. Our analysis predicts that there are at least 34 protein-coding regions in these genomes that were not apparent in earlier annotation efforts. These include 17 single-exon genes, three new exons of previously identified genes, a newly identified four-exon gene for a lectin-like protein (in rat cytomegalovirus), and 10 probable frameshift extensions of previously annotated genes. This expanded set of candidate genes provides an additional basis for investigation in cytomegalovirus biology and pathogenesis.

Amino Acid Sequence↗