Highly repetitive sequences and characteristics of genomic DNA in unicellular cyanobacterial strains

Abstract Microcystis aeruginosa (Synechocystis ) is a unicellular cyanobacterium that performs oxygenic photosynthesis. We found two novel sets of repetitive sequences, A (REP-A) and B (REP-B), on the M. aeruginosa K-81 genomic DNA, which consisted of distinct motifs of tandem repeated sequences located in the up- and downstream regions of the orf1 structural gene, respectively. Genomic Southern hybridization revealed multicopies of REP-A and -B on the genome. Furthermore, genomic Southern blots of cyanobacteria species with the REP-A and -B probes revealed that different hybridization signals appeared on the genomic DNAs of all 12 Microcystis strains, but no signal appeared on those of Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, and Anabaena sp. PCC 7120.     

Typing of Verotoxins by DNA Colony Hybridization with Poly- and Oligonucleotide Probes,a Bead-Enzyme-Linked Immunosorbent Assay,and Polymerase Chain Reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.     

Long-Term Acceptance of Major Histocompatibility Complex-Mismatched Cardiac Allograft Induced by a Low Dose of CTLA4IgM Plus FK506

The immunosuppressant FK506 prolongs allograft survival. However, at therapeutic doses it has significant side effects. A fusion protein consisting of the extracellular portion of CTLA4 and the Fc portion of human IgG (CTLA4IgG) also prolongs allograft survival, but large doses of CTLA4IgG are required for the induction of cardiac allograft acceptance. Therefore, we constructed a pentameric form of a new CTLA4 fusion protein, CTLA4IgM. We tested whether low doses of CTLA4IgG or CTLA4IgM in combination with subtherapeutic doses of FK506 can prolong allograft survival in a synergistic fashion. C57BL/6 (H-2^b) neonatal hearts were transplanted to CBA/J (H-2^k) mice in a heterotopic, nonvascularized cardiac allograft model. The findings demonstrate that a combination of low doses of FK506 plus a pentameric form of CTLA4Ig, CTLA4IgM, leads to significant graft survival, while a combination of FK506 plus CTLA4IgG does not.     

Cloning and characterization of the rice CatA catalase gene, a homologue of the maize Cat3 gene

We isolated and sequenced a genomic clone (CatA) encoding CAT-A catalase, a homologue of the maize catalase isozyme 3 (CAT-3) from rice (Oryza sativa L.). The 5-upstream non-coding region had very low similarity with the maize Cat3 gene and possible cis elements and sequence motifs in the maize Cat3 gene were not evident, except for TATA and CAAT motifs. Several sequence motifs found in the promoters of plant seed-specific genes were identified in the 5-upstream non-coding region of the CatA gene. Northern blotting showed that the CatA gene is expressed at high levels in seeds during early development and also in young seedlings. Methyl viologen (paraquat) resulted in the 3-fold induction of the CatA gene in the leaves of young seedlings, whereas abscisic acid, wounding, salicylic acid, and hydrogen peroxide had no or only slight effects.The 1.9 kb 5-upstream fragment (–1559 to +342) of the CatA gene was fused with the Escherichia coli -glucuronidase (GUS) gene and introduced by electroporation into protoplasts prepared from rice suspension-cultured cells, then the transient expression of the GUS gene was examined. Deletion analysis of this chimeric gene suggested that a weak silencer is located in the region between –1564 to –699. Abscisic acid (ABA) at a final concentration of 10^–6 M doubled GUS activity in protoplasts electroporated with the chimeric DNAs having 1.9 to 1.2 kb 5-upstream regions. A sequence highly similar to the Sph box, a motif found in genes modulated by ABA, was found at –266 to –254. Deletion of this region however, did not eliminate the responsiveness to ABA. Expression of the chimeric gene in the protoplasts was not enhanced by stress such as low and high temperature, hydrogen peroxide, methyl viologen, salicylic acid, elicitor, and UV light.The chimeric CatA-GUS plasmid DNAs amplified in the methylation-positive strain, E. coli DH5, showed GUS gene activities, whereas all the chimeric DNAs amplified in the methylation-deficient E. coli JM 110 were completely inactive in the presence or absence of ABA in the culture medium. DNA methylation, especially of either one or both of the deoxyadenosines at the two GATC motifs (one in the first exon and the other in the first intron of the rice CatA gene), appeared to be responsible for the CatA promoter activity identified in the transient assay.author for corresondenceThe nucleotide sequence data reported will appear in the DDBJ EMBL and GenBank Nucleotide Sequence Databases under the accession number D29966.     

Genomic Analysis of a New Mammalian Distal-less Gene: Dlx7

We have cloned a new Dlx gene (Dlx7) from human and mouse that may represent the mammalian orthologue of the newt geneNvHBox-5.The homeodomains of these genes are highly similar to all other vertebrate Dlx genes, and regions of similarity also exist between mammalian Dlx7 and a subset of vertebrate Dlx genes downstream of the homeodomain. The sequence divergence between human and mouse Dlx7 in these regions is greater than that predicted from comparisons of other vertebrate Dlx genes, however, and there is little sequence similarity upstream of the homeodomain both between these two genes and with other Dlx genes. We present evidence for alternative splicing of mouseDlx7upstream of the homeodomain that may account for some of this divergence. We have mapped humanDLX7distal to the 5′ end of the HOXB cluster at an estimated distance of between 1 and 2 Mb by FISH. Both the human and the mouse Dlx7 are shown to be closely linked to Dlx3 in a convergently transcribed orientation. These mapping results support the possibility that vertebrate distal-less genes have been duplicated in concert with the Hox clusters.     

Sarcoplasmic reticulum calsequestrins: Structural and functional properties

Calsequestrin is the major Ca²⁺-binding protein localized in the terminal cisternae of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle cells. Calsequestrin has been purified and cloned from both skeletal and cardiac muscle in mammalian, amphibian, and avian species. Two different calsequestrin gene products namely cardiac and fast have been identified. Fast and cardiac calsequestrin isoforms have a highly acidic amino acid composition. The amino acid composition of the cardiac form is very similar to the skeletal form except for the carboxyl terminal region of the protein which possess variable length of acidic residues and two phosphorylation sites. Circular dichroism and NMR studies have shown that calsequestrin increases its -helical content and the intrinsic fluorescence upon binding of Ca²⁺. Calsequestrin binds Ca²⁺ with high-capacity and with moderate affinity and it functions as a Ca²⁺ storage protein in the lumen of the SR. Calsequestrin has been found to be associated with the Ca²⁺ release channel protein complex of the SR through protein-protein interactions. The human and rabbit fast calsequestrin genes have been cloned. The fast gene is skeletal muscle specific and transcribed at different rates in fast and slow skeletal muscle but not in cardiac muscle. We have recently cloned the rabbit cardiac calsequestrin gene. Heart expresses exclusively the cardiac calsquestrin gene. This gene is also expressed in slow skeletal muscle. No change in calsequestrin mRNA expression has been detected in animal models of cardiac hypertrophy and in failing human heart.     

A multifunctional expression vector for an anti-HIV-1 ribozyme that produces a 5'- and 3'-trimmed trans-acting ribozyme, targeted against HIV-1 RNA, and cis-acting ribozymes that are designed to bind to and thereby sequester trans-activator proteins such as Tat and Rev.

We previously constructed a multiribozyme expression vector by combining cis- and trans-acting ribozymes and we showed that several ribozymes, each directed against a different target in the HIV genome and acting independently in a 'shotgun' manner, markedly increased the efficiency of cleavage of HIV RNA in vitro [Ohkawa et al., Proc. Natl Acad. Sci. USA 90, 11302 (1993)]. However, the cis-acting ribozymes that had trimmed the 5' and 3' ends of each trans-acting ribozyme were designed merely to await for degradation by RNases when they were used in vivo. Since several trans-activator proteins are essential for viral replication of HIV-1, we wondered whether a decoy function could be coupled with the cleavage activity of ribozymes. We therefore introduced the TAR or the RRE sequence into the stem II region of each cis-acting ribozyme. When the activity of each resulting cis-acting ribozyme that had been endowed with the decoy function was examined in vitro, it was found to retain almost full trimming activity. Moreover, cis-acting ribozymes with either the TAR or the RRE sequence were shown to be able to trap Tat or Rev protein successfully. It is, therefore, possible to endow the stem II region with a specific protein-binding function without the loss of ribozyme function. Thus, cis-acting ribozymes, endowed with the decoy function, can first trim the 5' and 3' ends of each trans-acting ribozyme and are then still available for trapping trans-activator proteins possibly prior to their degradation by RNases when they are to be used in vivo. Furthermore, it is also expected that the reduction in production of HIV RNA that is achieved by sequestering the trans-activator proteins might provide the trans-acting ribozymes, targeted to HIV RNA, with a better chance of eliminating the remaining HIV RNA.     

Effects of calf breed on milk production and other economic traits of Holstein dams

Production of purebred or crossbred feeder calves for beef, especially HolsteinxJapanese-Black (HxJB) and Japanese-Black (JB), from dairy cattle using artificial insemination or embryo transfer have been used widely in Japan. However, dairy farmers feel uneasy about the effects of calf breed on the economic traits of dams. In this study, those effects were investigated in 798 Holstein heifers bearing Holstein, HxJB, JB or other breed calves. The results of the least-squares ANOVA indicated the effects of calf breed to be significant for gestation length (P<0.01) and calf birth weight (P<0.01) but not for milk yield, fat yield, protein yield, peak yield, day of peak, number of artificial inseminations per pregnancy or days open. Frequency of dystocia was lower in dams bearing HxJB calves than in those bearing Holstein calves (P<0.05). There were no significant differences for frequencies of still birth, retained placenta and subsequent pregnancy. The present data suggest that the effects of calf breed do not place a serious problem on the economic traits of Holstein dams. In conclusion, it is indicated that the production of JB and HxJB feeder calves from Holstein dams does not result in a decrease in dam productivity.     

Conformation of (2→1)-β-d-fructan in aqueous solution

The conformation and dilute solution properties of (2→1)-β-d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (M_w) from 1.49 × 10⁴ to 5.29 × 10⁶ were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M_w < 4 × 10⁴, the intrinsic viscosity [η] varies with M_w^0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M_w > 10⁵, [η] exhibits an unusually weak dependence on M_w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.