Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain

Two overlapping genomic clones containing the fibroin light-chain (Fib-L)-encoding gene (Fib-L) were obtained from the cosmid library of the silkworm, Bombyx mori J-139, by hybridization with the Fib-L cDNA clone. Sequencing of the 14.6-kb region revealed that Fib-L was 13472 bp long containing seven exons, and that the gene contained a large first intron which occupied about 60% of the gene. Comparison of restriction patterns of the J-139 Fib-L with those of eight other B. mori breeds producing normal-level fibroin demonstrated that considerable restriction-fragment length polymorphisms were present in regions containing the first intron and the 3′-flanking sequence. However, sizes of the Fib-L mRNA and the Fib-L polypeptide were very similar among the nine breeds tested, suggesting that the exon sequences and the splice signals were all well conserved. 5′-Flanking regions of Fib-L and the fibroin heavy-chain (Fib-H)-encoding gene (Fib-H) compared in this study contained three 18-30-bp sequences of high similarity and many 8-10-bp common elements, six of which coincided with the binding sites of homeodomain proteins. Gel retardation assays with the nuclear extracts of the posterior and middle silk glands suggested that protein factors present in the posterior silk-gland nuclei could bind to a set of those common upstream elements.     

The lipoprotein lipase-encoding human gene: sequence from intron-6 to intron-9 and presence in intron-7 of a 40-million-year-old Alu sequence.

The complete nucleotide sequence of the 3877-bp segment spanning the 3′ region of intron-6 to the 5′ region of intron-9 of the human lipoprotein lipase (LPL)-encoding ten-exon gene, LPL, is reported. An Alu repeat present in intron-7 was found by sequence analysis to belong to the 40–55-million-year-old Alu-Se subclass.     

Spatial regulation of SpMTA metallothionein gene expression in sea urchin embryos by a regulatory cassette in intron 1

The SpMTA metallothionein (MT) gene of the sea urchin Strongylocentrotus purpuratus is restricted in its expression to the aboral ectoderm in gastrulae and pluteus larvae. The proximal 1.6 kb of the 5′-flanking region together with the 1.12-kb first intron of the SpMTA gene are sufficient for its correct cell-type specific expression in transgenic embryos. This restricted spatial expression is largely eliminated by deletion of an interior 405-bp region in the intron. Within this region is a 295-bp, genomically repetitive, transposon-like segment (Nemer et al., 1993), containing several sequence motifs highly homologous to posited regulatory elements in the promoters of other genes (Thiebaud et al., 1990). The P3A and P5 sites in this apparent regulatory cassette were shown through competition to bind with relatively high affinities the same nuclear factors, bound by their counterpart sites in the CyIIIa actin promoter.     

The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori

We have previously reported that Bmdsx, a homologue of the sex-determining gene, doublesex (dsx), was found to be sex-specifically expressed in various tissues at larval, pupal, and adult stages in the silkworm, Bombyx mori, and was alternatively spliced to yield male- and female-specific mRNAs. To reveal sex-specific differences in splicing patterns of Bmdsx pre-mRNA, the genomic sequence was determined and compared with male- and female-specific Bmdsx cDNA sequences. The open reading frame (ORF) consisted of five exons. Exons 3 and 4 were specifically incorporated into the female type of Bmdsx mRNA. On the other hand, exon 2 was spliced to exon 5 to produce the male type mRNA of Bmdsx. As in the case of Drosophila dsx, the OD2 domain was separated by a female-specific intron into sex-independent and sex-dependent regions. Sex-specific splicing occurred in equivalent positions in the Drosophila dsx gene. However, unlike Drosophila dsx, the female-specific introns showed no weak 3′ splice sites, and the TRA/TRA-2 binding site related sequences were not found in the female-specific exon, nor even in any other regions of the Bmdsx gene. Moreover, an in vitro splicing reaction consisting of HeLa cell nuclear extracts showed that the female-type of Bmdsx mRNA represented the default splicing. These findings suggest that the structural features of the sex-specific splicing patterns of Bmdsx pre-mRNA are similar to those of Drosophila dsx but the regulation of sex-specific alternative splicing of Bmdsx pre-mRNA is different.     

Cloning, expression and sequence analysis of the SphI restriction-modification system

SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5′-GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM. The complete sphIR was finally cloned using a two-step process. PCR was used to isolate the 3′ end of sphIR from a library. The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E. coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM). The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems. The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E. coli.     

The chorion genes of the medfly. II. DNA sequence evolution of the autosomal chorion genes s18, s15, s19 and s16 in Diptera

We present a total of approximately 15 kb of DNA sequences, encompassing four chorion genes Ccs18, Ccs15, Ccs19, Cc16 and their flanking DNA in the medfly C. capitata. Comparison of coding regions, introns and intergenic sequences in five Dipteran species, D. melanogaster, D. subobscura, D. virilis, D. grimshawi and C. capitata documented an extensive divergence in introns and coding regions, but few well conserved elements in the proximal 5′ flanking regions in all species. These elements are related to conserved regulatory features of three of the genes, including tissue- and temporal regulation. In the fourth, gene s15, significant alterations in the 5′ flanking region may be responsible for its changed temporal regulation in C. capitata. One long intergenic sequence, located in the distal 5′ flanking region of gene s18, is homologous to ACE3, a major amplification control element and contains an 80-bp A/T-rich sequence, known to stimulate strong binding of the origin recognition complex (ORC) in D. melanogaster. Analysis of the nucleotide composition of all chorion genes in C. capitata and D. melanogaster showed that C. capitata exhibit less biased representation of synonymous codons than does D. melanogaster.     

Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution

Antheraea pernyi silk fibroin fibers were dissolved by aqueous lithium thiocyanate to obtain regenerated A. pernyi silk fibroin solution. By means of circular dichroism, ¹³C NMR and Raman spectroscopy, the molecular conformation of regenerated A. pernyi silk fibroin in aqueous solution was investigated. The relationship of environmental factors and sol–gel transformation behavior of regenerated A. pernyi silk fibroin was also studied. The molecular conformations of regenerated A. pernyi silk fibroin mainly were -helix and random coil in solution. There also existed a little β-sheet conformation. It was obviously different with Bombyx mori silk fibroin, whose molecular conformation in solution was only random coil but no -helix existence. With the increase of temperature and solution concentration and with the decrease of solution pH value, the gelation velocity of regenerated A. pernyi silk fibroin solution increased. Especially, it showed that A. pernyi silk fibroin was more sensitive to temperature than B. mori silk fibroin during the sol–gel transformation. The velocity increased obviously when the temperature was above 30 °C. During the sol–gel transformation, the molecular conformation of regenerated A. pernyi silk fibroin changed from random coil to β-sheet structure. The results of these studies provided important insight into the preparation of new biomaterials by silk fibroin protein.     

Essential role of duplications of short motif sequences in the genomic evolution of Bombyx mori

Summary The Bombyx fibroin gene has a discrete mosaic structure of various repetitive sequences, which may have evolved through various repeating arrangements. Detailed sequence analysis of the fibroin gene containing coding and noncoding regions revealed that the whole sequence could be arranged as an array of short repetitive sequences. A portion of the intron of the fibroin gene is one of interspersed repetitive elements. We cloned a 1.5-kb DNA fragment of the Bombyx genome that contains interspersed elements homologous to the intron sequence. Sequence comparison between the intron and the 1.5-kb fragment shows that partial duplication has frequently occurred in evolutionary progress, and the resultant repetitive blocks of short motif sequences are abundant in the genome. These facts suggest that tandem duplication of the short motif sequence is an important rearrangement in genomic evolution of the fibroin gene. Offprint requests to: S. Ichimura     

Unique features in the mitochondrial D-loop region of the European seabass Dicentrarchus labrax

We have cloned and sequenced the displacement-loop (D-loop) region of the mitochondrial DNA (mtDNA) from the European seabass Dicentrarchus labrax (Dl). This sequencing revealed the presence of four tandemly repeated elements (R1, R2, R3 and R4); the individual variation in mtDNA total length is entirely accounted for by their variable number. The individuals examined also possessed an imperfect copy of one of the tandem repeats (ΨR2). At least one termination-associated sequence (TAS) is present in each of the repeats and in two copies 5′ upstream from the tandem array as well. The alignment of the Dl D-loop region with D-loop sequences from four other Teleosts and one Chondrosteus showed the Dl sequence to be larger than that of other fish. The extraordinary length of the D1 D-loop sequence is also due to the 5′ and 3′ regions that are flanking the tandem array, the largest ones to date analyzed in fish. In this study, we also report the unique organization and localization of putative TAS and conserved-sequence block (CSB) elements, and the presence of a conserved 218-bp sequence in the D1 D-loop region.