Integrons found in different locations have identical 5' ends but variable 3' ends.

The positions of the outer boundaries of the 5'- and 3'-conserved segment sequences of integrons found at several different locations have been determined. The position of the 5' end of the 5'-conserved segment is the same for six independently located integrons, In1 (R46), In2 (Tn21), In3 (R388), In4 (Tn1696), In5 (pSCH884), and In0 (pVS1). However, the extent of the 3'-conserved segment differs in each integron. The sequences of In2 and In0 diverge first from the conserved sequence, and their divergence point corresponds to the 3'-conserved segment endpoint defined previously (H.W. Stokes and R.M. Hall, Mol. Microbiol. 3:1669-1683, 1989), which now represents the endpoint of a 359-base deletion in In0 and In2. The sequence identity in In3, In1, In4, and In5 extends beyond this point, but each sequence diverges from the conserved sequence at a different point within a short region. Insertions of IS6100 were identified adjacent to the end of the conserved region in In1 and 123 bases beyond the divergence point of In4. These 123 bases are identical to the sequence found at the mer end of the 11.2-kb insertion in Tn21 but are inverted. In5 and In0 are bounded by the same 25-base inverted repeat that bounds the 11.2-kb insert in Tn21, and this insert now corresponds to In2. However, while In0, In2, and In5 have features characteristic of transposable elements, differences in the structures of these three integrons and the absence of evidence of mobility currently preclude the identification of all of the sequences associated with a functional transposon of this type.     

Integrons: Novel DNA elements which capture genes by site-specific recombination

Integrons are unusual DNA elements which include a gene encoding a site-specific DNA recombinase, a DNA integrase, and an adjacent site at which a wide variety of antibiotic resistance and other genes are found as inserts. One or more genes can be found in the insert region, but each gene is part of an independent gene cassette. The inserted genes are expressed from a promoter in the conserved sequences located 5 to the genes, and integrons are thus natural expression vectors. A model for gene insertion in which circular gene cassettes are inserted individually via a single site-specific recombination event has been proposed and verified experimentally. The gene cassettes include a gene coding region and, at the 3 end of the gene an imperfect inverted repeat, a 59-base element. The 59-base elements are a diverse family of elements which function as sites recognized by the DNA integrase. Site-specific insertion of individual genes thus represents a further mechanism which contributes to the evolution of the genomes of Gram-negative bacteria and their plasmids and transposons.Members of the most studied class of integrons, which include thesulI gene in the conserved sequences, are believed to be mobile DNA elements on the basis that they are found in many independent locations, and a discrete boundary is found at the outer end of the 5-conserved segment. However, the length of the 3-conserved segment is variable in the integrons examined to date, and it is likely that this variability has arisen as the result of insertion and deletion events. Though the true extent of the 3-conserved segment remains to be determined, it seems likely that these integrons are mobile DNA elements. The second known class of integrons comprises members of the Tn7 transposon family.     

Southern stingray (Dasyatis americana) feeding on lancelets (Branchiostoma floridae)

A southern stingray from the shallow sand flats of Tampa Bay, Florida, had its gut filled almost exclusively with lancelets. The absence of small lancelets from the gut contents indicated a pharyngeal sieving mechanism by the ray.     

Human alpha 3(VI) collagen gene. Characterization of exons coding for the amino-terminal globular domain and alternative splicing in normal and tumor cells

We recently reported the isolation and sequencing of human cDNA clones corresponding to the alpha 3 chain of type VI collagen (Chu, M.-L., Zhang, R.-Z., Pan, T.-c., Stokes, D., Conway, D., Kuo, H.-J., Glanville, R., Mayer, U., Mann, K., Deutzmann, R., and Timpl, R. (1990) EMBO J. 9, 385-393). The study indicates that the amino-terminal globular domain of the alpha 3(VI) chain consists of nine repetitive subdomains of approximately 200 amino acid residues (N1-N9) and the gene appeared to undergo alternative splicing since some clones lacked regions encoding the N9 and part of the N3 subdomains. In the present study, we report the exon structure for the region encoding the amino-terminal globular domain of the human alpha 3(VI) chain. The nine repetitive subdomains are encoded by 10 exons spanning 26 kilobase pairs of genomic DNA. Eight of the repetitive subdomains (N2-N9) were found to be encoded by separate exons of approximately 600 base pairs each. The only exception is the N1 subdomain which is encoded by two exons of 417 and 146 base pairs. Characterization of the exon/intron structure showed that the cDNA variants were the result of splicing out of exon 9 (encoding the N9 subdomain) and part of exon 3 (encoding the N3 subdomain). Nuclease S1 analysis and the polymerase chain reaction demonstrated that exon 7 (N7 subdomain) was also subject to alternative splicing in normal skin fibroblasts. Examination of these splicing events by nuclease S1 analysis in normal fibroblasts, three different human tumor cell lines, and several human tissues showed that splicing out of exon 9 is much more efficient in normal as compared to tumor cells.     

The integron In1 in plasmid R46 includes two copies of the oxa2 gene cassette.

The sequence of the insert region of the integron In1 found in the IncN plasmid R46 was completed. The insert region is 2929 bases long and includes four gene cassettes, two of which are identical copies of the oxa2 gene cassette flanking an aadA1 cassette. The fourth cassette encodes an open reading frame orfD. From comparison of these data with published maps and sequences it is argued that the integrons found in the IncN plasmids pCU1 and R1767 and in the transposon Tn2410 are closely related to In1 from R46. Both site-specific gene insertion and recA-dependent recombination are likely to have contributed to the evolution of these integrons.     

Monoclonal antibodies against the ruminal bacterium Selenomonas ruminantium.

Monoclonal antibodies were raised against whole cells of two different strains of Selenomonas ruminantium and tested for specificity and sensitivity in immunofluorescence and enzyme-linked immunosorbent assay procedures. Species-specific and strain-specific antibodies were identified, and reactive antigens were demonstrated in solubilized cell wall extracts of S. ruminantium. A monoclonal antibody-based solid-phase immunoassay was established to quantify S. ruminantium in cultures or samples from the rumen, and this had a sensitivity of 0.01 to 0.02% from 10(7) cells. For at least one strain, the extent of antibody reaction varied depending upon the stage of bacterial growth. Antigen characterization by immunoblotting shows that monoclonal antibodies raised against two different strains of S. ruminantium reacted with the same antigen on each strain. For one strain, an additional antigen reacted with both monoclonal antibodies. In the appropriate assay, these monoclonal antibodies may have advantages over gene probes, both in speed and sensitivity, for bacterial quantification studies.     

Enzyme histochemistry of the mesocoxal muscles of Periplaneta americana

Histochemical techniques have been employed to characterize enzymatic activity in the mesocoxal muscles of the cockroach, Periplaneta americana. Through our studies of the enzymes myosin-ATPase, NADH reductase, succinic dehydrogenase (SDH), and lactic dehydrogenase (LDH), we were able to classify fibers within these muscles according to criteria established for muscle fibers of vertebrates. Many of the mesocoxal muscles possess two different and distinct populations of fibers, whereas the remaining muscles are homogeneous with respect to their constituent fibers. The data presented here indicate biochemical heterogeneity for muscles of differing structural and functional features and possible neurotrophic influences upon oxidative enzymes and myosin-ATPase isozymes.