Genomic analysis of synaptotagmin genes.

I used TBLASTn to probe DNA sequence databases with a consensus peptide sequence corresponding to the most highly conserved region of the rodent synaptotagmin (Syt) gene family, which is within the C2B domain. I found human homologues for all known rodent genes, and found six further human genomic loci which encode potential family members. I found eight potential family members in Caenorhabditis elegans, six in Drosophila melanogaster, and four in Arabidopsis thaliana. The C. elegans Syt1 homologue uniquely encodes two alternative C2B exons, one or the other of which is expressed at a time. Comparison of the genomic structures of the Syt genes makes clear the different phylogenies of the different subgroups. Knowledge of the genomic structures will aid the systematic investigation of alternative splicing in Syt genes.

Alternative splicing of synaptotagmins involving transmembrane exon skipping.

The synaptotagmin gene family currently includes 12 members. Analysis of the three known genomic synaptotagmin sequences reveals conserved exon-intron patterns which delineate the synaptotagmin structural domains. We used expressed sequence tag, reverse transcription PCR and RNAse protection assay analysis of synaptotagmin messenger RNAs to demonstrate the occurrence of alternative splicing events involving a number of exons. Exon-skipped messages where transmembrane sequences have been removed or altered were found to be abundantly expressed by synaptotagmins 1, 4, 6 and 7. Although the expression of most synaptotagmins predominates in neural tissue, we find that by contrast, synaptotagmin 6 is more abundant in thymus.

Conversion of SB 203580-insensitive MAP kinase family members to drug-sensitive forms by a single amino-acid substitution.

BACKGROUND: Specific inhibitors of protein kinases have great therapeutic potential, but the molecular basis underlying their specificity is only poorly understood. We have investigated the drug SB 203580 which belongs to a class of pyridinyl imidazoles that inhibits the stress-activated protein (SAP) kinases SAPK2a/p38 and SAPK2b/p38 beta 2 but not other mitogen-activated protein kinase family members. Like inhibitors of other protein kinases, SB 203580 binds in the ATP-binding pocket of SAPK2a/p38. RESULTS: The SAP kinases SAPK1 gamma/JNK1, SAPK3 and SAPK4 are not inhibited by SB 203580, because they have methionine in the position equivalent to Thr106 in the ATP-binding region of SAPK2a/p38 and SAPK2b/p38 beta 2. Using site-directed mutagenesis of five SAP kinases and the type I and type II TGF beta receptors, we have established that for a protein kinase to be inhibited by SB 203580, the sidechain of this residue must be no larger than that of threonine. Sensitivity to inhibition by SB 203580 is greatly enhanced when the sidechain is even smaller, as in serine, alanine or glycine. Thus, the type I TGF beta receptor, which has serine at the position equivalent to Thr106 of SAPK2a/p38 and SAPK2b/p38 beta 2, is inhibited by SB 203580. CONCLUSIONS: These findings explain how drugs that target the ATP-binding site can inhibit protein kinases specifically, and show that the presence of threonine or a smaller amino acid at the position equivalent to Thr106 of SAPK2a/p38 and SAPK2b/p38 beta 2 is diagnostic of whether a protein kinase is sensitive to the pyridinyl imidazole class of inhibitor.

Aberrant intracellular targeting and cell cycle-dependent phosphorylation of emerin contribute to the Emery-Dreifuss muscular dystrophy phenotype.

The product of the X-linked Emery-Dreifuss muscular dystrophy gene is a protein called emerin, which is localized to the nuclear membrane. We have expressed full-length recombinant human emerin in an in vitro coupled reticulocyte system; it has a molecular mass of 34 kDa, inserts into microsomes in a type II orientation, and does not exhibit any N-linked glycosylation or cleavage event. Affinity-purified human emerin antiserum cross-reacts with the in vitro-expressed emerin and with a 34 kDa band present in a wide range of human tissue samples. Expression and subcellular distribution of emerin were studied in lymphoblastoid cell lines established from four patients with Emery-Dreifuss muscular dystrophy containing different mutations in the emerin gene. Emerin protein was detected in two of these patients by immunoblotting. In striking contrast to wild-type emerin, which was localized to the nuclear fraction and was insoluble in non-ionic detergents and high salt, emerin from these two patients exhibited a more random subcellular localization and increased solubility. On the basis of the mutations present in these patients, it would appear that emerin possesses two non-overlapping nuclear envelope targeting sequences. We have also demonstrated that emerin can occur in four different phosphorylated forms, three of which appear to be associated with the cell cycle. The mutant forms of emerin taken from the two patients exhibited aberrant cell cycle-dependent phosphorylated forms. This data suggests that for emerin to function normally it must be correctly localized, retained at the nuclear membrane and phosphorylated by cell cycle-mediated events.

Activation of the novel stress-activated protein kinase SAPK4 by cytokines and cellular stresses is mediated by SKK3 (MKK6); comparison of its substrate specificity with that of other SAP kinases.

A cDNA was cloned that encodes human stress-activated protein kinase-4 (SAPK4), a novel MAP kinase family member whose amino acid sequence is approximately 60% identical to that of the other three SAP kinases which contain a TGY motif in their activation domain. The mRNA encoding SAPK4 was found to be widely distributed in human tissues. When expressed in KB cells, SAPK4 was activated in response to cellular stresses and pro-inflammatory cytokines, in a manner similar to other SAPKs. SAPK4 was activated in vitro by SKK3 (also called MKK6) or when co-transfected with SKK3 into COS cells. SKK3 was the only activator of SAPK4 that was induced when KB cells were exposed to a cellular stress or stimulated with interleukin-1. These findings indicate that SKK3 mediates the activation of SAPK4. The substrate specificity of SAPK4 in vitro was similar to that of SAPK3. Both enzymes phosphorylated the transcription factors ATF2, Elk-1 and SAP-1 at similar rates, but were far less effective than SAPK2a (also called RK/p38) or SAPK2b (also called p38beta) in activating MAPKAP kinase-2 and MAPKAP kinase-3. Unlike SAPK1 (also called JNK), SAPK3 and SAPK4 did not phosphorylate the activation domain of c-Jun. Unlike SAPK2a and SAPK2b, SAPK4 and SAPK3 were not inhibited by the drugs SB 203580 and SB 202190. Our results suggest that cellular functions previously attributed to SAPK1 and/or SAPK2 may be mediated by SAPK3 or SAPK4.

Human synaptotagmin V (SYT5): sequence, genomic structure, and chromosomal location.

We have determined the sequence, genomic structure, and chromosomal location of the human synaptotagmin V (SYTV) gene. The human SYTV gene encodes a 386-amino-acid product which is 91% identical to rat Syt V. The human SYTV open reading frame is interrupted by seven introns which can be alternatively spliced. Human SYTV was found to lie very close to SYTIII on chromosome 19q13.4 by PCR analysis of somatic cell hybrid DNA and by DNA hybridization to arrayed cosmids of the chromosome 19 metric physical map. This provides the first report of linked synaptotagmin genes.

SAP kinase-3, a new member of the family of mammalian stress-activated protein kinases.

Stress-activated protein kinases are MAP kinase homologues that are activated by cellular stresses, bacterial endotoxin and inflammatory cytokines. They are activated by a dual threonine/tyrosine phosphorylation within a TPY sequence in the case of stress-activated protein kinase-1 and its isoforms (also called JNKs) or a TGY sequence in the case of stress-activated protein kinase-2 and its isoforms (also called p38, p40, RK, CSBPs, XMpk2 and Mxi2). Here we report the cloning and sequencing of a new protein kinase from rat with a TGY sequence in the activation domain. This stress-activated protein kinase-3 is 60% identical to mouse stress-activated protein kinase-2 and 45% identical to HOG1 from Saccharomyces cerevisiae. Transcripts encoding stress-activated protein kinase-3 are widely expressed, with high levels in skeletal muscle.

The DNA sequence of human herpesvirus-6: structure, coding content, and genome evolution.

The complete DNA sequence was determined for strain U1102 of human herpesvirus-6, a CD4+ T-lymphotropic virus with disease associations in immunodeficient settings and a possible complicating factor in AIDS. The genome is 159,321 bp in size, has a base composition of 43% G + C, and contains 119 open reading frames. The overall structure is 143 kb bounded by 8 kb of direct repeats, DRL (left) and DRR (right), containing 0.35 kb of terminal and junctional arrays of human telomere-like simple repeats. Since eight open reading frames are duplicated in the repeats, six span repetitive elements and three are spliced, the genome is considered to contain 102 separate genes likely to encode protein. The genes are arranged colinearly with those in the genome of the previously sequenced betaherpesvirus, human cytomegalovirus, and has a distinct arrangement of conserved genes relative to the sequenced gammaherpesviruses, herpesvirus saimiri and Epstein-Barr virus, and the alphaherpesviruses, equine herpesvirus-1, varicella-zoster virus, and herpes simplex virus. Comparisons of predicted amino acid sequences allowed the functions of many human herpesvirus-6 encoded proteins to be assigned and showed the closest relationship in overall number and similarity to human cytomegalovirus products, with approximately 67% homologous proteins as compared to the 21% identified in all herpesviruses. The features of the conserved genes and their relative order suggested a general scheme for divergence among these herpesvirus lineages. In addition to the "core" conserved genes, the genome contains four distinct gene families which may be involved in immune evasion and persistence in immune cells: two have similarity to the "chemokine" chemotactic/proinflammatory family of cytokines, one to their peptide G-protein-coupled receptors, and a fourth to the immunoglobulin superfamily.

Synaptotagmin V: a novel synaptotagmin isoform expressed in rat brain.

Regulated Ca(2+)-dependent release of transmitters from synaptic vesicles is an important characteristic of chemical neurotransmission. Synaptotagmins are abundant synaptic vesicle transmembrane proteins that probably function as Ca2+ sensors. Molecular cloning has identified four different synaptotagmin isoforms in mammals. We report here the cloning and sequencing of a novel isoform of 386 amino acids. Synaptotagmin V is 54% identical in sequence to synaptotagmin I and possesses all the domains that characterise this multigene family. It is expressed at high levels in rat brain, but not in spinal cord or a number of peripheral non-neuronal tissues.

Primary structure of the herpesvirus saimiri genome.

This report describes the complete nucleotide sequence of the genome of herpesvirus saimiri, the prototype of gammaherpesvirus subgroup 2 (rhadinoviruses). The unique low-G + C-content DNA region has 112,930 bp with an average base composition of 34.5% G + C and is flanked by about 35 noncoding high-G + C-content DNA repeats of 1,444 bp (70.8% G + C) in tandem orientation. We identified 76 major open reading frames and a set of seven U-RNA genes for a total of 83 potential genes. The genes are closely arranged, with only a few regions of sizable noncoding sequences. For 60 of the predicted proteins, homologous sequences are found in other herpesviruses. Genes conserved between herpesvirus saimiri and Epstein-Barr virus (gammaherpesvirus subgroup 1) show that their genomes are generally collinear, although conserved gene blocks are separated by unique genes that appear to determine the particular phenotype of these viruses. Several deduced protein sequences of herpesvirus saimiri without counterparts in most of the other sequenced herpesviruses exhibited significant homology with cellular proteins of known function. These include thymidylate synthase, dihydrofolate reductase, complement control proteins, the cell surface antigen CD59, cyclins, and G protein-coupled receptors. Searching for functional protein motifs revealed that the virus may encode a cytosine-specific methylase and a tyrosine-specific protein kinase. Several herpesvirus saimiri genes are potential candidates to cooperate with the gene for saimiri transformation-associated protein of subgroup A (STP-A) in T-lymphocyte growth stimulation.

A survey of expressed genes in Caenorhabditis elegans.

As an adjunct to the genomic sequencing of Caenorhabditis elegans, we have investigated a representative cDNA library of 1,517 clones. A single sequence read has been obtained from the 5' end of each clone, allowing its characterization with respect to the public databases, and the clones are being localized on the genome map. The result is the identification of about 1,200 of the estimated 15,000 genes of C. elegans. More than 30% of the inferred protein sequences have significant similarity to existing sequences in the databases, providing a route towards in vivo analysis of known genes in the nematode. These clones also provide material for assessing the accuracy of predicted exons and splicing patterns and will lead to a more accurate estimate of the total number of genes in the organism than has hitherto been available.

The genome of human herpesvirus 6: maps of unit-length and concatemeric genomes for nine restriction endonucleases.

More than 50 fragments resulting from complete digestion of the DNA of human herpesvirus 6 (HHV-6, strain U1102) with BamHI, EcoRI, HindIII, KpnI, NruI, SalI or SmaI have been isolated as clones in M13, plasmid, cosmid and lambda vectors. Using these clones, maps have been constructed for the fragments produced by nine restriction enzymes from unit-length virus genomes and from their concatemeric precursors. The unit-length genome is a linear, double-stranded molecule of 161.5 kbp composed of a central segment of a largely unique sequence of 141 kbp (U) with a sequence of 10 kbp duplicated in the same orientation at both 'left' and 'right' genomic termini (i.e. 'left' and 'right' copies of the direct repeat; DRL and DRR). Adopting as standard an orientation in which the major capsid protein gene is 'left' of the gene for alkaline exonuclease, then the 'right' genome termini and DRL. U junctions occur close to or within repetitive (GGGTTA)n sequences. Repetitions of short sequence motifs are present in at least two other regions of the genome. One of these regions consists of a simple repeat (TC/G) of approximately 1.5 kbp in length and is unstable as clones in bacterial vectors. The second region is stably maintained in such vectors and consists of a tandem array of at least 25 copies of a 110 bp sequence containing a single KpnI site. Comparisons of fragments arising from unit-length DNA with those from virus DNA from the nuclei of infected cells have shown that the concatemeric junctions in intracellular DNA contain head-to-tail dimers of the terminal duplications (i.e. ...U1.DRR1.DRL2.U2...). The gross structure established here for the genome from the U1102 isolate of HHV-6 resembles closely that suggested by Pellett and his colleagues for the Z29 isolate and differs from that of the five previously characterized human herpesviruses. This structure of HHV-6 DNA bears a superficial resemblance to that proposed for DNA from channel catfish virus and equine cytomegalovirus.

Human herpesvirus 6 is closely related to human cytomegalovirus.

A sequence of 21,858 base pairs from the genome of human herpesvirus 6 (HHV-6) strain U1102 is presented. The sequence has a mean composition of 41% G + C, and the observed frequency of CpG dinucleotides is close to that predicted from this mononucleotide composition. The sequence contains 17 complete open reading frames (ORFs) and part of another at the 5' end of the sequence. The predicted protein products of two of these ORFs have no recognizable homologs in the genomes of other sequenced human herpesviruses (i.e., Epstein-Barr virus [EBV], human cytomegalovirus [HCMV], herpes simplex virus [HSV], and varicella-zoster virus [VZV]). However, the products of nine other ORFs are clearly homologous to a set of genes that is conserved in all other sequenced herpesviruses, including homologs of the alkaline exonuclease, the phosphotransferase, the spliced ORF, and the major capsid protein genes. Measurements of similarity between these homologous sequences showed that HHV-6 is clearly most closely related to HCMV. The degree of relatedness between HHV-6 and HCMV was commensurate with that observed in comparisons between HSV and VZV or EBV and herpesvirus saimiri and significantly greater than its relatedness to EBV, HSV, or VZV. In addition, the gene for the major capsid protein and its 5' neighbor are reoriented with respect to the spliced ORFs in the genomes of both HHV-6 and HCMV relative to the organization observed in EBV, HSV, and VZV. Three ORFs in HHV-6 have recognizable homologs only in the genome of HCMV. Despite differences in gross composition and size, we conclude that the genomes of HHV-6 and HCMV are closely related.