Enhancement of translation by the epsilon element is independent of the sequence of the 460 region of 16S rRNA

The epsilon enhancer element is a pyrimidine-rich sequence that increases expression of T7 gene 10 and a number of Escherichia coli mRNAs during initiation of translation and inhibits expression of the recF mRNA during elongation. Based on its complementarity to the 460 region of 16S rRNA, it has been proposed that epsilon exerts its enhancer activity by base pairing to this complementary rRNA sequence. We have tested this model of enhancer action by constructing mutations in the 460 region of 16S rRNA and examining expression of epsilon-containing CAT reporter genes and recF–lacZ fusions in strains expressing the mutant rRNAs. Replacement of the 460 E.coli stem–loop with that of Salmonella enterica serovar Typhimurium or a stem–loop containing a reversal of all 8 bp in the helical region produced fully functional rRNAs with no apparent effect on cell growth or expression of any epsilon-containing mRNA. Our experiments confirm the reported effects of the epsilon elements on gene expression but show that these effects are independent of the sequence of the 460 region of 16S rRNA, indicating that epsilon–rRNA base pairing does not occur.

Sequence-tagged microsatellite profiling (STMP): a rapid technique for developing SSR markers

We describe a technique, sequence-tagged microsatellite profiling (STMP), to rapidly generate large numbers of simple sequence repeat (SSR) markers from genomic or cDNA. This technique eliminates the need for library screening to identify SSR-containing clones and provides an ∼25-fold increase in sequencing throughput compared to traditional methods. STMP generates short but characteristic nucleotide sequence tags for fragments that are present within a pool of SSR amplicons. These tags are then ligated together to form concatemers for cloning and sequencing. The analysis of thousands of tags gives rise to a representational profile of the abundance and frequency of SSRs within the DNA pool, from which low copy sequences can be identified. As each tag contains sufficient nucleotide sequence for primer design, their conversion into PCR primers allows the amplification of corresponding full-length fragments from the pool of SSR amplicons. These fragments permit the full characterisation of a SSR locus and provide flanking sequence for the development of a microsatellite marker. Alternatively, sequence tag primers can be used to directly amplify corresponding SSR loci from genomic DNA, thereby reducing the cost of developing a microsatellite marker to the synthesis of just one sequence-specific primer. We demonstrate the utility of STMP by the development of SSR markers in bread wheat.

Complete genome sequence of an M1 strain of Streptococcus pyogenes

The 1,852,442-bp sequence of an M1 strain of Streptococcus pyogenes, a Gram-positive pathogen, has been determined and contains 1,752 predicted protein-encoding genes. Approximately one-third of these genes have no identifiable function, with the remainder falling into previously characterized categories of known microbial function. Consistent with the observation that S. pyogenes is responsible for a wider variety of human disease than any other bacterial species, more than 40 putative virulence-associated genes have been identified. Additional genes have been identified that encode proteins likely associated with microbial “molecular mimicry” of host characteristics and involved in rheumatic fever or acute glomerulonephritis. The complete or partial sequence of four different bacteriophage genomes is also present, with each containing genes for one or more previously undiscovered superantigen-like proteins. These prophage-associated genes encode at least six potential virulence factors, emphasizing the importance of bacteriophages in horizontal gene transfer and a possible mechanism for generating new strains with increased pathogenic potential.

Sequence-Specific Interaction between the Disintegrin Domain of Mouse ADAM 3 and Murine Eggs: Role of β1 Integrin-associated Proteins CD9, CD81, and CD98

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-α6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-α6 mAb, or by mAbs against either the αv or β3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other β1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg β1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface “tetraspan web” facilitates fertilization and that it may do so by fostering ADAM–integrin interactions.

Sequence conservation at human and mouse orthologous common fragile regions, FRA3B/FHIT and Fra14A2/Fhit

It has been suggested that delayed DNA replication underlies fragility at common human fragile sites, but specific sequences responsible for expression of these inducible fragile sites have not been identified. One approach to identify such cis-acting sequences within the large nonexonic regions of fragile sites would be to identify conserved functional elements within orthologous fragile sites by interspecies sequence comparison. This study describes a comparison of orthologous fragile regions, the human FRA3B/FHIT and the murine Fra14A2/Fhit locus. We sequenced over 600 kbp of the mouse Fra14A2, covering the region orthologous to the fragile epicenter of FRA3B, and determined the Fhit deletion break points in a mouse kidney cancer cell line (RENCA). The murine Fra14A2 locus, like the human FRA3B, was characterized by a high AT content. Alignment of the two sequences showed that this fragile region was stable in evolution despite its susceptibility to mitotic recombination on inhibition of DNA replication. There were also several unusual highly conserved regions (HCRs). The positions of predicted matrix attachment regions (MARs), possibly related to replication origins, were not conserved. Of known fragile region landmarks, five cancer cell break points, one viral integration site, and one aphidicolin break cluster were located within or near HCRs. Thus, comparison of orthologous fragile regions has identified highly conserved sequences with possible functional roles in maintenance of fragility.

Excitation–contraction coupling is unaffected by drastic alteration of the sequence surrounding residues L720–L764 of the α1S II-III loop

The II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) α1S subunit is responsible for bidirectional-signaling interactions with the ryanodine receptor (RyR1): transmitting an orthograde, excitation–contraction (EC) coupling signal to RyR1 and receiving a retrograde, current-enhancing signal from RyR1. Previously, several reports argued for the importance of two distinct regions of the skeletal II-III loop (residues R681–L690 and residues L720–Q765, respectively), claiming for each a key function in DHPR–RyR1 communication. To address whether residues 720–765 of the II-III loop are sufficient to enable skeletal-type (Ca2+ entry-independent) EC coupling and retrograde interaction with RyR1, we constructed a green fluorescent protein (GFP)-tagged chimera (GFP-SkLM) having rabbit skeletal (Sk) DHPR sequence except for a II-III loop (L) from the DHPR of the house fly, Musca domestica (M). The Musca II-III loop (75% dissimilarity to α1S) has no similarity to α1S in the regions R681–L690 and L720–Q765. GFP-SkLM expressed in dysgenic myotubes (which lack endogenous α1S subunits) was unable to restore EC coupling and displayed strongly reduced Ca2+ current densities despite normal surface expression levels and correct triad targeting (colocalization with RyR1). Introducing rabbit α1S residues L720–L764 into the Musca II-III loop of GFP-SkLM (substitution for Musca DHPR residues E724–T755) completely restored bidirectional coupling, indicating its dependence on α1S loop residues 720–764 but its independence from other regions of the loop. Thus, 45 α1S-residues embedded in a very dissimilar background are sufficient to restore bidirectional coupling, indicating that these residues may be a site of a protein–protein interaction required for bidirectional coupling.

Highly specific protein sequence motifs for genome analysis

We present a method for discovering conserved sequence motifs from families of aligned protein sequences. The method has been implemented as a computer program called emotif (http://motif.stanford.edu/emotif). Given an aligned set of protein sequences, emotif generates a set of motifs with a wide range of specificities and sensitivities. emotif also can generate motifs that describe possible subfamilies of a protein superfamily. A disjunction of such motifs often can represent the entire superfamily with high specificity and sensitivity. We have used emotif to generate sets of motifs from all 7,000 protein alignments in the blocks and prints databases. The resulting database, called identify (http://motif.stanford.edu/identify), contains more than 50,000 motifs. For each alignment, the database contains several motifs having a probability of matching a false positive that range from 10−10 to 10−5. Highly specific motifs are well suited for searching entire proteomes, while generating very few false predictions. identify assigns biological functions to 25–30% of all proteins encoded by the Saccharomyces cerevisiae genome and by several bacterial genomes. In particular, identify assigned functions to 172 of proteins of unknown function in the yeast genome.

Precise sequence complementarity between yeast chromosome ends and two classes of just-subtelomeric sequences

The terminal regions (last 20 kb) of Saccharomyces cerevisiae chromosomes universally contain blocks of precise sequence similarity to other chromosome terminal regions. The left and right terminal regions are distinct in the sense that the sequence similarities between them are reverse complements. Direct sequence similarity occurs between the left terminal regions and also between the right terminal regions, but not between any left ends and right ends. With minor exceptions the relationships range from 80% to 100% match within blocks. The regions of similarity are composites of familiar and unfamiliar repeated sequences as well as what could be considered “single-copy” (or better “two-copy”) sequences. All terminal regions were compared with all other chromosomes, forward and reverse complement, and 768 comparisons are diagrammed. It appears there has been an extensive history of sequence exchange or copying between terminal regions. The subtelomeric sequences fall into two classes. Seventeen of the chromosome ends terminate with the Y′ repeat, while 15 end with the 800-nt “X2” repeats just adjacent to the telomerase simple repeats. The just-subterminal repeats are very similar to each other except that chromosome 1 right end is more divergent.

A unified statistical framework for sequence comparison and structure comparison

We present an approach for assessing the significance of sequence and structure comparisons by using nearly identical statistical formalisms for both sequence and structure. Doing so involves an all-vs.-all comparison of protein domains [taken here from the Structural Classification of Proteins (scop) database] and then fitting a simple distribution function to the observed scores. By using this distribution, we can attach a statistical significance to each comparison score in the form of a P value, the probability that a better score would occur by chance. As expected, we find that the scores for sequence matching follow an extreme-value distribution. The agreement, moreover, between the P values that we derive from this distribution and those reported by standard programs (e.g., blast and fasta validates our approach. Structure comparison scores also follow an extreme-value distribution when the statistics are expressed in terms of a structural alignment score (essentially the sum of reciprocated distances between aligned atoms minus gap penalties). We find that the traditional metric of structural similarity, the rms deviation in atom positions after fitting aligned atoms, follows a different distribution of scores and does not perform as well as the structural alignment score. Comparison of the sequence and structure statistics for pairs of proteins known to be related distantly shows that structural comparison is able to detect approximately twice as many distant relationships as sequence comparison at the same error rate. The comparison also indicates that there are very few pairs with significant similarity in terms of sequence but not structure whereas many pairs have significant similarity in terms of structure but not sequence.

Comparative organization of cattle chromosome 5 revealed by comparative mapping by annotation and sequence similarity and radiation hybrid mapping

A whole genome cattle-hamster radiation hybrid cell panel was used to construct a map of 54 markers located on bovine chromosome 5 (BTA5). Of the 54 markers, 34 are microsatellites selected from the cattle linkage map and 20 are genes. Among the 20 mapped genes, 10 are new assignments that were made by using the comparative mapping by annotation and sequence similarity strategy. A LOD-3 radiation hybrid framework map consisting of 21 markers was constructed. The relatively low retention frequency of markers on this chromosome (19%) prevented unambiguous ordering of the other 33 markers. The length of the map is 398.7 cR, corresponding to a ratio of ≈2.8 cR5,000/cM. Type I genes were binned for comparison of gene order among cattle, humans, and mice. Multiple internal rearrangements within conserved syntenic groups were apparent upon comparison of gene order on BTA5 and HSA12 and HSA22. A similarly high number of rearrangements were observed between BTA5 and MMU6, MMU10, and MMU15. The detailed comparative map of BTA5 should facilitate identification of genes affecting economically important traits that have been mapped to this chromosome and should contribute to our understanding of mammalian chromosome evolution.

Mutations within a furin consensus sequence block proteolytic release of ectodysplasin-A and cause X-linked hypohidrotic ectodermal dysplasia

X-linked hypohidrotic ectodermal dysplasia (XLHED) is a heritable disorder of the ED-1 gene disrupting the morphogenesis of ectodermal structures. The ED-1 gene product, ectodysplasin-A (EDA), is a tumor necrosis factor (TNF) family member and is synthesized as a membrane-anchored precursor protein with the TNF core motif located in the C-terminal domain. The stalk region of EDA contains the sequence -Arg-Val-Arg-Arg156-Asn-Lys-Arg159-, representing overlapping consensus cleavage sites (Arg-X-Lys/Arg-Arg↓) for the proprotein convertase furin. Missense mutations in four of the five basic residues within this sequence account for ≈20% of all known XLHED cases, with mutations occurring most frequently at Arg156, which is shared by the two consensus furin sites. These analyses suggest that cleavage at the furin site(s) in the stalk region is required for the EDA-mediated cell-to-cell signaling that regulates the morphogenesis of ectodermal appendages. Here we show that the 50-kDa EDA parent molecule is cleaved at -Arg156Asn-Lys-Arg159↓- to release the soluble C-terminal fragment containing the TNF core domain. This cleavage appears to be catalyzed by furin, as release of the TNF domain was blocked either by expression of the furin inhibitor α1-PDX or by expression of EDA in furin-deficient LoVo cells. These results demonstrate that mutation of a functional furin cleavage site in a developmental signaling molecule is a basis for human disease (XLHED) and raise the possibility that furin cleavage may regulate the ability of EDA to act as a juxtacrine or paracrine factor.