Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid.

The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti contains three replicons: pSymA, pSymB, and the chromosome. We report here the complete 1,354,226-nt sequence of pSymA. In addition to a large fraction of the genes known to be specifically involved in symbiosis, pSymA contains genes likely to be involved in nitrogen and carbon metabolism, transport, stress, and resistance responses, and other functions that give S. meliloti an advantage in its specialized niche.

The composite genome of the legume symbiont Sinorhizobium meliloti.

The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.

Population genetic implications from sequence variation in four Y chromosome genes.

Some insight into human evolution has been gained from the sequencing of four Y chromosome genes. Primary genomic sequencing determined gene SMCY to be composed of 27 exons that comprise 4,620 bp of coding sequence. The unfinished sequencing of the 5' portion of gene UTY1 was completed by primer walking, and a total of 20 exons were found. By using denaturing HPLC, these two genes, as well as DBY and DFFRY, were screened for polymorphic sites in 53-72 representatives of the five continents. A total of 98 variants were found, yielding nucleotide diversity estimates of 2.45 x 10(-5), 5. 07 x 10(-5), and 8.54 x 10(-5) for the coding regions of SMCY, DFFRY, and UTY1, respectively, with no variant having been observed in DBY. In agreement with most autosomal genes, diversity estimates for the noncoding regions were about 2- to 3-fold higher and ranged from 9. 16 x 10(-5) to 14.2 x 10(-5) for the four genes. Analysis of the frequencies of derived alleles for all four genes showed that they more closely fit the expectation of a Luria-Delbrück distribution than a distribution expected under a constant population size model, providing evidence for exponential population growth. Pairwise nucleotide mismatch distributions date the occurrence of population expansion to approximately 28,000 years ago. This estimate is in accord with the spread of Aurignacian technology and the disappearance of the Neanderthals.

Comparative genomes of Chlamydia pneumoniae and C. trachomatis.

Chlamydia are obligate intracellular eubacteria that are phylogenetically separated from other bacterial divisions. C. trachomatis and C. pneumoniae are both pathogens of humans but differ in their tissue tropism and spectrum of diseases. C. pneumoniae is a newly recognized species of Chlamydia that is a natural pathogen of humans, and causes pneumonia and bronchitis. In the United States, approximately 10% of pneumonia cases and 5% of bronchitis cases are attributed to C. pneumoniae infection. Chronic disease may result following respiratory-acquired infection, such as reactive airway disease, adult-onset asthma and potentially lung cancer. In addition, C. pneumoniae infection has been associated with atherosclerosis. C. trachomatis infection causes trachoma, an ocular infection that leads to blindness, and sexually transmitted diseases such as pelvic inflammatory disease, chronic pelvic pain, ectopic pregnancy and epididymitis. Although relatively little is known about C. trachomatis biology, even less is known concerning C. pneumoniae. Comparison of the C. pneumoniae genome with the C. trachomatis genome will provide an understanding of the common biological processes required for infection and survival in mammalian cells. Genomic differences are implicated in the unique properties that differentiate the two species in disease spectrum. Analysis of the 1,230,230-nt C. pneumoniae genome revealed 214 protein-coding sequences not found in C. trachomatis, most without homologues to other known sequences. Prominent comparative findings include expansion of a novel family of 21 sequence-variant outer-membrane proteins, conservation of a type-III secretion virulence system, three serine/threonine protein kinases and a pair of parologous phospholipase-D-like proteins, additional purine and biotin biosynthetic capability, a homologue for aromatic amino acid (tryptophan) hydroxylase and the loss of tryptophan biosynthesis genes.

The mouse mahogany locus encodes a transmembrane form of human attractin.

Agouti protein and agouti-related protein are homologous paracrine signalling molecules that normally regulate hair colour and body weight, respectively, by antagonizing signalling through melanocortin receptors. Expression of Agouti is normally limited to the skin, but rare alleles from which Agouti is expressed ubiquitously, such as lethal yellow, have pleiotropic effects that include a yellow coat, obesity, increased linear growth, and immune defects. The mahogany (mg) mutation suppresses the effects of lethal yellow on pigmentation and body weight, and results of our previous genetic studies place mg downstream of transcription of Agouti but upstream of melanocortin receptors. Here we use positional cloning to identify a candidate gene for mahogany, Mgca. The predicted protein encoded by Mgca is a 1,428-amino-acid, single-transmembrane-domain protein that is expressed in many tissues, including pigment cells and the hypothalamus. The extracellular domain of the Mgca protein is the orthologue of human attractin, a circulating molecule produced by activated T cells that has been implicated in immune-cell interactions. These observations provide new insight into the regulation of energy metabolism and indicate a molecular basis for crosstalk between melanocortin-receptor signalling and immune function.

The nucleotide sequence of Saccharomyces cerevisiae chromosome V.

Here we report the sequence of 569,202 base pairs of Saccharomyces cerevisiae chromosome V. Analysis of the sequence revealed a centromere, two telomeres and 271 open reading frames (ORFs) plus 13 tRNAs and four small nuclear RNAs. There are two Tyl transposable elements, each of which contains an ORF (included in the count of 271). Of the ORFs, 78 (29%) are new, 81 (30%) have potential homologues in the public databases, and 112 (41%) are previously characterized yeast genes.

Quantitative trait locus analysis of susceptibility to diet-induced atherosclerosis in recombinant inbred mice.

Quantitative trait locus (QTL) analysis is a statistical method that can be applied to identify loci making a significant impact on a phenotype. For the phenotype of susceptibility to diet-induced atherosclerosis in the mouse, we have studied four quantitative traits: area of aortic fatty streaks and serum concentrations of high-density lipoprotein-bound cholesterol (HDL-cholesterol), apolipoprotein A-I, and apolipoprotein A-II (apo A-II). QTL analysis revealed a significant locus on chromosome 1 distal impacting serum apo A-II concentration on a high-fat diet and serum HDL-cholesterol concentration on a chow diet. This locus is presumably Apoa-2, the structural gene for apo A-II. QTL analysis of aortic fatty streaks failed to reveal a significant locus.

Rapid screening of a human genomic library in yeast artificial chromosomes for single-copy sequences.

A yeast artificial chromosome (YAC) library in Saccharomyces cerevisiae consisting of 30,000 clones with an average insert size of 0.1 megabase pair of human DNA has been generated from primary fibroblast DNA. A YAC vector was modified to enable the recovery of both ends of a human DNA insert in plasmids in Escherichia coli and to confer G418 resistance to mammalian cells. A rapid method for yeast colony hybridization was used that exploits the ability of yeast spheroplasts to regenerate in a thin layer of calcium alginate. This method permits direct replica plating and processing of colonies from the primary transformation plate to nitrocellulose filters. Yeast colony hybridization conditions have been established to identify, within a YAC library of human genomic DNA, artificial chromosomes with homology to human DNA probes of unique single-copy sequence. An artificial chromosome with a 0.1-megabase-pair insert from the human Xq28 region has been identified by hybridization to a DNA probe that detects a unique sequence near the 3' end of the factor VIII gene.

Isomerization of the UL region of varicella-zoster virus DNA.

Varicella-zoster virus (VZV) DNA exists principally as two isomers. Despite the presence of inverted repeats bounding the long sequence region, the unique long sequence, UL, is found in one (prototype) orientation in 95-98% of VZV DNA molecules and in the inverted orientation in only 2-5% of the molecules. In searching for an explanation for this disparity, we superinfected VZV-infected cells with herpes simplex virus type 1 or pseudorabies virus. Neither superinfecting virus produced a measurable change in the frequency of isomerization of the VZV DNA long sequence region.

Latent herpesvirus infections of neurons in guinea pigs and humans.

Latent herpes simplex virus (HSV) infection of the trigeminal ganglion of guinea pigs and latent varicella-zoster virus (VZV) infection of the trigeminal ganglion of humans were studied by in situ nucleic acid hybridization. Guinea pig trigeminal ganglia were removed during the period of viral latency (four to five weeks after corneal inoculation of HSV), and human ganglia were removed at autopsy. Radiolabeled HSV and VZV DNAs were used to probe ganglion tissue sections for viral-specified RNA. Hybridization detected only over neurons was present in 46 percent of ganglia from 22 latently infected guinea pigs and from 33 percent of ganglia from 10 human subjects. These results support the conclusion that some viral transcription occurred during HSV and VZV latency.

Hybridization between a repeated region of herpes simplex virus type 1 DNA containing the sequence [GGC]n and heterodisperse cellular DNA and RNA.

A small DNA fragment containing the simple sequence [GGC]10 from the long repeat of herpes simplex virus type 1 (HSV-1) DNA hybridized to cellular DNA and polyadenylated RNA from different mammalian species. The number and intensity of blot hybridization signals were increased in human compared with rodent and simian nucleic acids. The hybridization was blocked specifically by human 28S ribosomal DNA, which shares only the GGC repeats with the herpes simplex virus DNA. These data indicate that GGC repeats were common components of cellular DNA and were expressed in mRNA. Blot hybridization analysis of viral RNA from the HSV-1 gene regions encompassing the GGC repeats revealed abundant stable mRNAs from portions of the virus genome not previously analyzed in detail and indicated that the viral GGC sequence was not expressed in stable cytoplasmic mRNA.

The immediate early proteins of varicella-zoster virus.

Stable, relatively high-titer varicella-zoster virus (VZV) stocks as well as a high-titer anti-VZV serum prepared in inbred guinea pigs have allowed the identification of VZV immediate early proteins using the classic inhibitor approach. VZV infection was initiated in the presence of cycloheximide. Following the removal of cycloheximide, actinomycin D and radiolabel were added. After the labeling period, extracts were immunoprecipitated with anti-VZV guinea pig serum and subjected to polyacrylamide gel electrophoresis and fluorography or autoradiography. Four immediate early proteins of mol wts 185,000, 69,000, 43,000, and 34,000 were identified. The largest three were phosphoproteins.

A simple repetitive sequence common to herpes simplex virus type 1 and human ribosomal DNAs.

The simple sequence GGC was tandemly repeated in herpes simplex virus type 1 DNA and human 28S rDNA and its mature 28 S rRNA transcript. The sequence homology was responsible for the observed hybridization between the two DNAs under high stringency blot hybridization conditions.

Sequences in the proximal IRL of herpes simplex virus DNA hybridize to human DNA.

A short segment of the herpes simplex virus (HSV) DNA proximal IRL region hybridized to two doublet bands of EcoRI-digested human cellular DNA. The hybridization was abolished neither by increasing stringency conditions nor by inclusion of guanine-rich DNA in the hybridization mixture. Thus, the hybridization appeared to represent authentic base sequence homology between HSV DNA and middle repetitive human DNA.

Polyadenylated, cytoplasmic transcripts of varicella-zoster virus.

Cytoplasmic RNA was isolated from varicella-zoster virus (VZV)-infected cells. By oligo(dT)-cellulose chromatography, the RNA was separated into polyadenylated, poly (A)+, and nonpolyadenylated, poly (A)-, fractions. RNA blot hybridization was employed to detect and map VZV transcripts. As VZV infection cannot be coordinated, cytoplasmic RNA was isolated from VZV-infected cells when the cells showed extensive cytopathology. Therefore, while the VZV transcripts represented heterogeneous temporal classes, it may be assumed that late VZV RNA predominated. At least 41, and as many as 67 (depending on DNA probe overlap), VZV polyadenylated transcripts have been identified. Preliminary evidence for the presence of two VZV-specific nonpolyadenylated, cytoplasmic transcripts was observed.

Errant processing and structural alterations of genomes present in a varicella-zoster virus vaccine.

Five minority populations of aberrant, varicella-zoster virus (VZV)-derived genomes were identified among the encapsidated DNAs obtained from the nuclear and cytoplasmic fractions of an in vitro infection initiated with a lyophilized sample of the BIKEN VZV vaccine (strain Oka). These were (i) VZV genomes, present within nuclear but not cytoplasmic viral capsids, which had been cleaved at a specific site within the short segment and which were, therefore, 3.15 megadaltons (approximately 4% of the VZV genome length) short of full length; (ii) highly deleted, repetitive VZV genomes which contained the errant cleavage site but not the usual VZV genome terminal sequences; (iii) VZV genomes into which multiples of 1 through 5 defective genome repeat units had been inserted into a homologous site; (iv) VZV genomes with additions of 0.1 or 0.18 megadaltons of DNA at both the terminal and internal ends of the short segment; and (v) VZV DNA which had lost the HindIII restriction site at map position 0.11.

Hybridization of herpes simplex virus DNA and human ribosomal DNA and RNA.

A small DNA segment from the inverted repeats at the termini of the unique long sequence region of herpes simplex virus DNA was found to hybridize with human 28 S ribosomal DNA and RNA but not 18 S ribosomal DNA and RNA. The hybridization occurred under stringent conditions and was not blocked by nucleic acids high in guanine plus cytosine content. These data strongly suggest that the hybridization represented authentic base sequence homology.

Variation in the structure of varicella-zoster virus DNA.

By analyzing the fine structure of varicella-zoster virus (VZV) DNA, a naturally occurring heterogeneity was found on the right end of VZV DNA, but no evidence of a true terminal repetition was uncovered. We were unable to confirm the report of Straus and co-workers (1981) that there is a relatively high frequency of circular VZV DNA in low-passage virus. On long-term cell passage, extensive heterogeneity appeared concomitant with the accumulation of apparently defective VZV DNA.

Specious hybridization between herpes simplex virus DNA and human cellular DNA.

Apparent hybridization between human cellular DNA and herpes simplex virus DNA was blocked by the presence of guanine-rich ribo- and deoxyribonucleic acid polymers. The data indicate that the apparent hybridization may very well be artifactual and not represent long stretches of authentic base sequence homology.