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Differential use of termination codons in ciliated protozoa.

Sequence analysis of genes in four species of ciliated protozoa and analysis of tRNAs in Tetrahymena has demonstrated that TAG and TAA encode glutamine or glutamic acid in these organisms and TGA is the only stop codon. Thus, it has generally been assumed that all ciliates use a nonuniversal genetic code in which TGA acts as the sole termination codon. We have sequenced the linear DNA molecules that carry an actin gene and a beta-tubulin gene from the ciliate Euplotes crassus. These genes are shown to use TAA as a termination codon based on homology to known actin and beta-tubulin gene sequences. In addition, we have sequenced a portion of the 3' terminus of the E. crassus H4 histone gene and show that it also uses TAA as a termination codon. These data indicate that the timing of genetic code changes in the ciliates must be reconsidered.

Actins

Changes in Connecticut hospital use rates: have small-area variations been affected?

This paper examines the extent to which changes (prospective payment, alternative delivery systems, etc.) in the hospital environment and the general decline in hospital days affect small-area variations in hospital use rates for 18 selected diagnoses in nine hospital service areas in Connecticut. After adjusting for coding changes between DRGs, we found that variation across the service areas did not, in general, differ for any one of the years 1981-86. In one instance (cardiac catheterization), however, we found that a DRG-specific change in knowledge and technology decreased the extent of small-area variation for that diagnosis.

Analysis of Variance

A chromosome 12 coding region is juxtaposed to the MYC protooncogene locus in a t(8;12)(q24;q22) translocation in a case of B-cell chronic lymphocytic leukemia.

We performed molecular cloning and sequencing of the breakpoints of a new chromosomal translocation involving the MYC protooncogene locus. This secondary t(8;12)(q24;q22) was associated with a primary t(11;14)(q13;q32) translocation in a case of B-cell chronic lymphocytic leukemia (CLL) in blastic transformation. In this leukemia, Northern blot and nuclease analyses SI showed that MYC was strongly expressed with initiation of the transcription at both the 5' and 3' promoters as observed in Burkitt's lymphomas; no coding change was observed in MYC putative regulatory sequences. The breakpoint on chromosome 8 mapped to the 3' end of the MYC locus, in a region containing a potential Z-DNA tract, and where we identified two DNase 1 hypersensitive sites. A rearranged MYC gene fragment was cloned and shown to contain chromosome 12 information by Southern blot analysis and by in situ hybridization. A genomic probe subcloned from the isolated region of the chromosome 12 recognized a 1.8 kb transcript in virtually all the tissues tested but a preferential expression of this new gene, which we termed BTG1 (for B-cell translocation gene 1) was observed in the CLL cells and in tissues of lymphoid origin. This chromosome 12 coding sequence is conserved in evolution and a transcript of similar size is present in murine tissues.

Animals

Alternative forms of a strain-specific neutralizing antigenic site on the Sindbis virus E2 glycoprotein.

Experiments with monoclonal antibodies raised against two laboratory strains of Sindbis virus, SB and SIN, suggested the existence of a strain-specific neutralizing antigenic site (E2-b) on the E2 glycoprotein. A comparison of monoclonal antibody binding patterns and E2 glycoprotein gene sequences of six laboratory strains distinguished three different configurations of E2-b that correlated with specific amino acid substitutions at position 216 of the E2 glycoprotein. Further study of neutralization escape mutants selected with E2-b-specific antibodies confirmed that amino acid 216 is a major determinant of the E2-b antigenic site. Eight of nine mutants showed a coding change at position 216. One neutralization escape mutation created a new glycosylation site at position 213 and resulted in an E2 protein with an altered migration rate in SDS-PAGE. The neutralization escape mutants studied included amino acid substitutions not found in the laboratory strains that revealed differing binding requirements for two E2-b-specific monoclonal antibodies. The E2-b site is contrasted with the E2-c neutralizing antigenic site described previously (R.A. Olmsted, W.J. Meyer, and R.E. Johnston, 1986, Virology 148, 245-254).

Amino Acid Sequence

Canine host range and a specific epitope map along with variant sequences in the capsid protein gene of canine parvovirus and related feline, mink, and raccoon parvoviruses.

Canine parvovirus (CPV) is a recently recognized pathogen of dogs that is similar to the long-recognized feline, mink, and raccoon parvoviruses. Relationships between the viruses determined from DNA sequences of the capsid protein genes of 10 virus isolates showed the CPV isolates to be closely related to the other viruses, although comprising a distinct group. No immediate ancestor of CPV was observed amongst the mink, cat, or raccoon viruses examined. Three different directly repeated sequences were present within the noncoding region downstream from the capsid protein genes. Analysis of recombinants between CPV and feline panleukopenia virus at restriction sites within the capsid protein genes mapped a CPV-specific neutralization epitope on the virus capsid, differences in the pH dependence of hemagglutination, and part of the determinant of canine host range between 59 and 64 genome map units (m.u.). Those differences were therefore the result of up to three nucleotide or predicted amino acid sequence differences in that region. A second region between 64 and 73 m.u., which may affect the viability of certain recombinant viruses, contained four nucleotide differences, one of which was a coding change.

Animals

Mapping specific functions in the capsid structure of canine parvovirus and feline panleukopenia virus using infectious plasmid clones.

DNA sequences between 0 and 98.8 genome map units (m.u.) from canine parvovirus (CPV) and feline panleukopenia virus (FPV) were cloned into plasmid vectors to form infectious molecular clones. Those plasmids were transfected into permissive cells and viruses recovered were shown to contain intact genomes, having regenerated the complete viral 5' ends up to 100 m.u. The viruses derived from the plasmids were compared to the original viruses, and shown to be indistinguishable in antigenic type, hemagglutination (HA) type and host range. The plasmid origin of the viruses was shown by preparing recombinant clones between CPV and FPV, and demonstrating the recombinant nature of the resulting viruses by restriction mapping and by sequencing viral DNA across the recombination sites. The sequences of our wild-type isolates CPV-d and FPV-b were completed, revealing 50 nucleotide sequence differences, of which 16 determined coding changes--5 in NS-1,2 in NS-2, and 9 in VP-2 protein. The sequences of the 5' ends (95.3-100 m.u.) of both viruses were also determined. Analysis of recombinant viruses mapped both CPV- and FPV-specific antigenic epitopes, the pH dependence of HA, and sequences affecting canine host range of the viruses within the VP-1 and VP-2 structural protein genes. Most of the specific changes were shown to be either on, or within one amino acid of, the surface of the virus capsid, indicating that the exposed surface of the parvovirus capsid plays an important role in determining a number of virus functions. The specific epitopes were affected by differences in a raised area on the capsid ("threefold spike"), while the pH dependence of HA difference was adjacent to a depression in the surface of the capsid at the twofold axis of symmetry.

Animals

Molecular characterization and expression of glycoprotein gene of Hantavirus R22 strain isolated from Rattus norvegicus in China.

A cDNA containing the complete open reading frame of the M genome segment of Hantavirus R22 strain isolated from Rattus norvegicus in China, was amplified by polymerase chain reaction (PCR), and then cloned. The M segment is 3656 nucleotides in length with a predicted region of 3402 bases encoding a precursor glycoprotein of 1134 amino acids subsequently processed into viral glycoproteins 1 and 2 (G1 and G2). A strain comparison between R22 and SR11 (isolated from a rat in Japan), and Hantaan 76-118 (isolated from Apodemus in Korea), and Hallnas B1 (isolated from a bank vole in Sweden) revealed 95%, 74%, and 53% homologies at the deduced amino acid sequence level respectively. This suggests that the rodent host species may be a more important determinant of genetic relationships than geographic proximity. Six potential asparagine linked glycosylation sites (five in G1 and one in G2) were identified, and among them all are conserved in SR11, five in Hantaan virus and four in Hallnas B1 virus. Although different degrees of homology exist among these four viruses at amino acid sequence level, more than 90% of the cysteine residues are conserved, suggesting that structural homology may be very strong between the Hantaviruses. Genetic differences in the M segment genome of R22 and SR11 viruses, within the same serotype viruses, were found as random coding changes; some limited to single amino acids, others in clusters. A recombinant vaccinia virus that contained the fully activated M segment cDNA of R22 was constructed. This recombinant virus expressed two glycoproteins G1 and G2 identical to R22 virus G1 and G2 in molecular weight, cleavage pattern and cellular immunofluorescent patterns.

Amino Acid Sequence

Chemical methods of protein synthesis and modification.

Chemical and recombinant methods have continued to complement one another in the synthesis of protein analogues. Chemical methods remain particularly valuable when non-coded modifications are to be introduced, although it has been accepted since the commercialization of semisynthetic human insulin that they can also be used effectively for coded changes, in certain cases. The main objective of all such operations is not methodological, but is the production of molecules for practical use and further study. This goal has been reached frequently by chemical means during the past year.

Base Sequence

Genetic modulation of antigen presentation by HLA-B27 molecules.

In studies of antigenic peptide presentation, we have found a healthy volunteer whose lymphoblastoid cells were unable to present three different virus-derived epitopes to cytotoxic T lymphocytes (CTL) despite expressing the correct restricting HLA-B27 molecules on the cell surface. B cell lines were established from other members of the donor's family, including individuals suffering from ankylosing spondylitis and related diseases, and were tested for their ability to function as target cells in the same assay. None of the eight B cell lines that expressed HLA-B27 presented a known peptide epitope to CTL. However, cells from a family member that expressed HLA-B8 could present an epitope peptide restricted by that molecule. The B27 molecule in this family proved to be the B2702 subtype on isoelectric focusing gels, appearing in exactly the same position as B2702 from other cell lines that did present the peptide. To exclude mutations resulting in noncharged amino acid substitutions, cDNA coding for B2702 was cloned from the proband's cell line and sequenced. No coding changes were found. The cloned cDNA was transfected into HLA-A- and B-negative HMy/C1R cells, and the B2702 molecules generated in this environment rendered these cells, after incubation with peptide, susceptible to lysis by peptide-specific CTL. These data are compatible with the presence of a factor(s), possibly HLA linked, interfering with antigen presentation by otherwise normal B2702 molecules in this family.

Antigen-Presenting Cells

Comparative Phylogenetics Reveal Clade-specific Drivers of Recombination Rate Evolution Across Vertebrates.

Meiotic recombination is an integral cellular process, required for the production of viable gametes. Recombination rate is a fundamental genomic parameter, modulating genomic responses to selection. Our increasingly detailed understanding of its molecular underpinnings raises the prospect that we can gain insight into trait divergence by examining the molecular evolution of recombination genes from a pathway perspective, as in mammals, where protein-coding changes in later stages of the recombination pathway are connected to divergence in intra-clade recombination rate. Here, we leverage increased availability of avian and teleost genomes to reconstruct the evolution of the recombination pathway across two additional vertebrate clades: birds, which have higher and more variable rates of recombination and similar divergence times to mammals, and teleost fish, which have much deeper divergence times. Rates of molecular evolution of recombination genes are highly correlated between vertebrate clades and significantly elevated compared to control panels, suggesting that they experience similar selective pressures. Avian recombination genes are significantly more likely to exhibit signatures of positive selection than other clades, unrestricted to later stages of the pathway. Signatures of positive selection in genes linked to recombination rate variation in mammalian populations and those with signatures of positive selection across the avian phylogeny are highly correlated. In contrast, teleost fish recombination genes have significantly less evidence of positive selection despite high intra-clade recombination rate variability. Gaining clade-specific understanding of patterns of variation in recombination genes can elucidate drivers of recombination rate and thus, factors influencing genetic diversity, selection efficacy, and species divergence.

Animals

Current peptic ulcer time trends. An epidemiological profile.

There has been speculation that peptic ulcer is a vanishing disease. To determine current time trends for peptic ulcers, we examined data from the National Center for Health Statistics and the Commission on Professional and Hospital Activities covering the period 1970 to 1985. For duodenal ulcer, hospitalizations for uncomplicated cases continue to decline, but there has been no decrease in cases with complications (such as perforations or hemorrhages). Hospitalizations for gastric ulcer are increasing, especially for cases with hemorrhage. Duodenal and gastric ulcer death rates have remained fairly constant since 1979. Site-unspecified peptic ulcer death rates doubled in 1979 and have remained at this rate through 1983. This increase can largely be attributed to coding changes made in the 9th revision of the International Classification of Diseases.

Duodenal Ulcer

QRS amplitudes, QTc intervals and ECG abnormalities in pheochromocytoma patients before, during and after treatment.

QRS amplitudes (S1 + R5), Minnesota Code ECG abnormalities and QTc intervals were analyzed in ECGs from 31 pheochromocytoma patients on admission to hospital, after 17 +/- 7.2 (SD) days of preoperative phenoxybenzamine therapy in a dosage of 148 +/- 45.0 (SD) mg/day and after a mean postoperative follow-up of 6 years. QRS voltage decreased significantly from 3.37 +/- 1.48 on admission, to 2.26 +/- 0.69 mV at postoperative follow-up (p less than 0.001). The prevalence of total Minnesota Code changes increased or was unchanged on the different occasions. Severe to moderate changes, however, became less prevalent, while mild changes increased. The QTc interval was 0.44 +/- 0.04 s on admission and 0.41 +/- 0.02 s at follow-up (p less than 0.01). No significant differences were observed during treatment with phenoxybenzamine. Pathological ECGs are common findings in patients with pheochromocytoma and significant improvements occur after surgery. Significant effects of phenoxybenzamine therapy were only found in patients with sustained hypertension.

Electrocardiography

Nucleotide sequence of a neurovirulent variant of the type 2 oral poliovirus vaccine.

Infectious cDNAs of the Sabin type 2 poliovirus vaccine virus and a vaccine-derived neurovirulent type 2 strain (P2/117) have been cloned in Escherichia coli. Nucleotide sequence analysis revealed that P2/117 differs from the vaccine strain by just 23 point mutations. Three occur in the 5' noncoding region. The remainder result in a total of 5 coding changes located in VP1, VP4, 2B, and 3D. The likely role of these mutations in the evolution to neurovirulence is discussed.

Animals

Mutational analysis of a virulence locus in the E2 glycoprotein gene of Sindbis virus.

The substitution of arginine for serine at position 114 of glycoprotein E2 in several biological and recombinant Sindbis virus mutants was shown previously to attenuate the virus for neonatal mice and also to accelerate virus penetration into BHK cells. To further examine the genetically linked effects on both virus penetration into cultured cells and pathogenesis in vivo, mutants containing each of 16 different amino acid coding changes at this position were generated by site-directed mutagenesis of a full-length cDNA clone of the Sindbis virus genome. Viable virus was recovered following transfection of RNA transcripts from 14 of the clones. Phenotypic analysis of these virus mutants revealed that specific amino acid residues affected either the pathogenesis or penetration phenotype independently or both phenotypes simultaneously. Thus, both the position of a mutation within the E2 sequence and the particular amino acid encoded at that position are important determinants of the mutant phenotypes.

Amino Acid Sequence

Moloney murine sarcoma virus MuSVts110 DNA: cloning, nucleotide sequence, and gene expression.

We have cloned Moloney murine sarcoma virus (MuSV) MuSVts110 DNA by assembly of polymerase chain reaction (PCR)-amplified segments of integrated viral DNA from infected NRK cells (6m2 cells) and determined its complete sequence. Previously, by direct sequencing of MuSVts110 RNA transcribed in 6m2 cells, we established that the thermosensitive RNA splicing phenotype uniquely characteristic of MuSVts110 results from a deletion of 1,487 nucleotides of progenitor MuSV-124 sequences. As anticipated, the sequence obtained in this study contained precisely this same deletion. In addition, several other unexpected sequence differences were found between MuSVts110 and MuSV-124. For example, in the noncoding region upstream of the gag gene, MuSVts110 DNA contained a 52-nucleotide tract typical of murine leukemia virus rather than MuSV-124, suggesting that MuSVts110 originated as a MuSV-helper murine leukemia virus recombinant during reverse transcription rather than from a straightforward deletion within MuSV-124. In addition, both MuSVts110 long terminal repeats contained head-to-tail duplications of eight nucleotides in the U3 region. Finally, seven single-nucleotide substitutions were found scattered throughout MuSVts110 DNA. Three of the nucleotide substitutions were in the gag gene, resulting in one coding change in p15 and one in p30. All of the remaining nucleotide changes were found in the noncoding region between the 5' long terminal repeat and the gag gene. In NIH 3T3 cells transfected with the cloned MuSVts110 DNA, the pattern of viral RNA expression conformed with that observed in cells infected with authentic MuSVts110 virus in that viral RNA splicing was 30 to 40% efficient at growth temperatures between 28 and 33 degrees C but reduced to trace levels above 37 degrees C.

3T3 Cells

Restricted use of fetal VH3 immunoglobulin genes by unselected B cells in the adult. Predominance of 56p1-like VH genes in common variable immunodeficiency.

The large VH3 family of human immunoglobulin genes is commonly used throughout B cell ontogeny. However, B cells of the fetus and certain autoantibody-producing clones are restricted to a recurrent subset of VH3 genes, and VH3 B cells are deficient in certain immunodeficiency diseases. In this study, we have sequenced a set of rearranged VH3 genes generated by genomic polymerase chain reaction (PCR) from normal adults and those with common variable immunodeficiency (CVI). In both groups, all cones were readily identifiable with the fetal VH3 subset, and were further distinguished by limited DH motifs and exclusive use of JH4. In CVI, the residual population of VH3 B cells were notable for predominant use of 56p1-like VH genes. All clones displayed sequence divergence (including somatic mutation) with evidence of strong selection against complementarity-determining region (CDR) coding change. A survey of other V gene families indicates that human V gene diversity may be restricted in general by germline mechanisms. These findings suggest that the expressed antibody repertoire in the human adult may be much smaller than anticipated, and selected by processes in part distinct from the paradigm of maximal antigen-binding diversity.

Antibody Diversity

Exon 7 Ncol restriction site within CYP21B (steroid 21-hydroxylase) is a normal polymorphism.

A point mutation within exon 7 producing an amino acid coding change and a recognition site for the endonuclease Ncol has been reported in the HLA-Bw47-linked CYP21A pseudogene and some mutant CYP21B (steroid 21-hydroxylase) genes of patients with congenital adrenal hyperplasia (CAH). Whether this mutation is deleterious was not demonstrated. We analyzed DNA from various subjects for the presence of the exon 7 Ncol site: group 1, 10 normal subjects; group 2, 11 patients with salt-losing CAH; and group 3, 18 members of an Amish pedigree in which 10 expressed HLA-Bw47 not linked to CAH. Southern blots of Ncol-digested genomic DNA which were hybridized with CYP21 cDNA showed that four subjects of group 1 had a heterozygous Ncol pattern. In group 2, seven patients had the Ncol site; two of them were homozygous for the site and had deletions of both CYP21B genes. The other five were heterozygous for the Ncol site, which was linked to a CYP21B deletion and a HLA-Bw47 haplotype. In group 3, no one exhibited the exon 7 Ncol site. To map the Ncol sites to CYP21A or CYP21B in the normal subjects, DNA from the four Ncol heterozygous subjects was double digested with Ncol and Mbol and hybridized with CYP21 cDNA. Ncol-Mbol fragments unique to CYP21A were identified in all four, but the smaller CYP21B-specific fragments were not detected. Their genomic DNA in the region of exon 7 (bases +1167 to +2058) was then amplified, cloned, and sequenced.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Hyperplasia, Congenital

Prioritization of causal genes from genome-wide association studies by Bayesian data integration across loci.

MOTIVATION: Genome-wide association studies (GWAS) have identified genetic variants, usually single-nucleotide polymorphisms (SNPs), associated with human traits, including disease and disease risk. These variants (or causal variants in linkage disequilibrium with them) usually affect the regulation or function of a nearby gene. A GWAS locus can span many genes, however, and prioritizing which gene or genes in a locus are most likely to be causal remains a challenge. Better prioritization and prediction of causal genes could reveal disease mechanisms and suggest interventions. RESULTS: We describe a new Bayesian method, termed SigNet for significance networks, that combines information both within and across loci to identify the most likely causal gene at each locus. The SigNet method builds on existing methods that focus on individual loci with evidence from gene distance and expression quantitative trait loci (eQTL) by sharing information across loci using protein-protein and gene regulatory interaction network data. In an application to cardiac electrophysiology with 226 GWAS loci, only 46 (20%) have within-locus evidence from Mendelian genes, protein-coding changes, or colocalization with eQTL signals. At the remaining 180 loci lacking functional information, SigNet selects 56 genes other than the minimum distance gene, equal to 31% of the information-poor loci and 25% of the GWAS loci overall. Assessment by pathway enrichment demonstrates improved performance by SigNet. Review of individual loci shows literature evidence for genes selected by SigNet, including PMP22 as a novel causal gene candidate.

Genome-Wide Association Study