DNA sequence of Nodulin-45 from Lupinus angustifolius

A partial cDNA clone encoding Lupinus angustifolius Nodulin-45 was isolated by differential hybridisation. A genomic clone was also isolated, from which the DNA sequence was obtained for the 5′ end of the gene (including 1.2 kb of 5′ upstream region). The upstream region includes putative cis-elements, found upstream of other nodulin genes. Southern analysis indicates the presence of several Nodulin-45-like sequences in the lupin genome. The Nodulin-45 protein has a putative N-terminal endoplasmic reticulum-type signal sequence and also contains a large glycine-rich repeat sequence. The cDNA sequence is highly homologous to a Nodulin-45 cDNA sequence from Lupinus luteus (Szczyglowski et al., Plant Sci., 65 (1989) 87–95), although major sequence rearrangements are apparent between the L. luteus and L. angustifolius cDNAs.     

Structure of the murine lactotransferrin gene is similar to the structure of other transferrin-encoding genes and shares a putative regulatory region with the murine myeloperoxidase gene

The structure and nucleotide sequence of the murine lactotransferrin-encoding gene (LTF) deduced partly by direct sequencing of genomic clones in the λ phage vector and partly by enzymatic amplification of genomic DNA segments primed with the oligodeoxyribonucleic primers homologous to the cDNA sequence. The λ phage clones contained the 5′ half of the gene corresponding to the first eight exons and an incomplete ninth exon interrupted by eight introns. Genomic clones corresponding to the 3′ half of the LTF gene could not be obtained on repeated attempts from two different mouse genomic libraries, suggesting the possible presence of unclonable sequences in this part of the gene. Hence, PCR was used to clone the rest of the gene. Four out of the presumed eight remaining introns were cloned along with the flanking exons using PCR. Comparison of the structure of the LTF gene with those of the two other known transferrin-encoding genes, human serum transferrin-encoding gene and chicken ovotransferrin-encoding gene reveals that all three genes have a very similar intron-exon distribution pattern. The hypothesis that the present-day transferrin-encoding genes have originated from duplication of a common ancestral gene is confirmed here at the gene level. An interesting finding is the identification of a region of shared nucleotides between the 5′ flanking regions of the murine LTF and myeloperoxidase-encoding genes, the two genes expressed specifically in neutrophilic granulocytes.     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus?

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

Tissue distribution, hormone regulation and evidence for a human homologue of the estrogen-inducible Xenopus laevis vitellogenin mRNA binding protein

17β-estradiol induces the synthesis of massive amounts of the hepatic mRNA encoding the Xenopus laevis egg yolk precursor protein, vitellogenin. Vitellogenin mRNA exhibits a half life of approx. 500 h when 17β-estradiol is present, and 16 h after removal of 17β-estradiol from the culture medium. We recently reported that Xenopus liver contains a protein, which is induced by 17β-estradiol and binds with a high degree of specificity to a binding site in a segment of the 3′-untranslated region (3′-UTR) of vitellogenin mRNA implicated in 17β-estradiol stabilization of vitellogenin mRNA. To determine if this mRNA binding protein was specific to this system, or if it was present elsewhere, and regulated by other steroids, we examined the tissue distribution and androgen regulation of this protein. Substantial amounts of the vitellogenin 3′-UTR binding protein were found in several Xenopus tissues including testis, ovary and muscle. In the absence of hormone treatment, lung and intestine contained minimal levels of the mRNA binding protein. Testosterone administration induced the vitellogenin 3′-UTR RNA binding protein in several tissues. Additionally, we found a homologous mRNA binding protein in MCF-7, human breast cancer cells. Although the MCF-7 cell protein was not induced by 17β-estradiol, the MCF-7 cell mRNA binding protein appears to be closely related to the Xenopus protein since: (i) the human and Xenopus proteins elicit gel shifted bands with the same electrophoretic mobility using the vitellogenin mRNA 3′-UTR binding site; (ii) The human and Xenopus proteins exhibit similar binding specificity for the vitellogenin 3′-UTR RNA binding site; and (iii) RNA from MCF-7 cells is at least as effective as RNA from control male Xenopus liver in blocking the binding of the Xenopus and human proteins to the vitellogenin mRNA 3′-UTR binding site. Its broad tissue distribution and regulation by both 17β-estradiol and testosterone suggests that this mRNA binding protein may play a significant role in steroid hormone regulation of mRNA metabolism in many vertebrate cells.     

Replication origin of the Bacillus subtilis chromosome determined by hybridization of the first-replicating DNA with cloned fragments from the replication origin region of the chromosome

The replication origin (ori) on the Bacillus subtilis genome was determined by the hybridization between the first-replicating DNA region and the cloned fragments from the ori region. The first-replicating DNA region was labeled specifically by [³H]thymidine in the presence of an inhibitor for DNA polymerase during a synchronous initiation of the chromosomal replication by germinating spores starved for thymine, and isolated by a sucrose density gradient centrifugation. Most of the labeled DNA molecules are small in size (up to 1000 bases long). The 45-kb ori region was cloned first in a λ Charon vector and then subcloned in pBR vectors. Restriction fragments from these cloned DNAs were purified by electrophoresis in agarose gels.

Only one region within the 45-kb ori region shows strong hybridization with the first-replicating DNA. Restriction fragments from this region were cloned in a phage M13 vector and separated into complementary strands. Hybridization of the labeled DNA with these cloned single-stranded fragments revealed that one site of the ori is located in each strand and they are some 2-kb apart from each other. Replication starts from these sites and proceeds inwards to pass each other.     

Efficient establishment of pig embryonic fibroblast cell lines with conditional expression of the simian vacuolating virus 40 large T fragment

The pig is an important animal for both agricultural and medical purposes. However, the number of pig-derived cell lines is relatively limited when compared with mouse- and human-derived lines. We established in this study a retroviral conditional expression system for the Simian vacuolating virus 40 large T fragment (SV40T) which allowed us to efficiently establish pig embryonic fibroblast cell lines. The established cell lines showed high levels of cell proliferation and resistance to cellular senescence. A chromosome analysis showed that 84% of the cells had the normal karyotype. Transient expression of the Cre recombinase allowed us to excise the SV40T fragment from the genome. The development of this research tool will enable us to quickly establish new cell lines derived from various animals.     

Sequence analysis of the 5′-flanking region of the gene encoding human N-acetylglucosaminyltransferase III

We have isolated and characterized the immediate (1651 bp) 5′-flanking region of the gene (GnT-III) encoding N-acetylglucosaminyltransferase III (GnT-III) from a human placental genomic library. Analysis of promoter elements shows a similarity to the 5′-flanking region of murine 1,4-galactosyltransferase. The sequence lacks obvious TATA elements and CCAAT boxes; however, putative regulatory sites, including 2 potential cAMP-response regulatory elements (CRE), 11 insulin-response element consensus sequences (IRE), 7 potential AP-2-binding sites, 2 SP1 consensus sequences (GC boxes) and 2 sequences similar to the half-palindromic glucocorticoid-responsive element (GRE), are present.     

Neolignans from Ocotea catharinensis

Bark, wood and leaves of Ocotea catharinensis contain respectively 10 (average yield 0.7%.), 15 (average yield 0.004%.) and one (yield 0.4%.) neolignans of the bicyclo[3.2.1]octanoid and the hydrobenzofuranoid structural types, including the new rel-(7S,8R,1′R,4′S,5′R,6′R)-Δ^8′-4′,6′-dihydroxy-5′-methoxy-3,4-methylenedioxy-3′-oxo-8.1′,7.5′-neolignan, (7S,8S)-Δ^{1′,3′,5′,8′}-5,3′,5′-trimethoxy-3,4-methylenedioxy-8.1′,7.O.6′,4.O.7′-neolignan, (7R,8S,1′R,3′R)-Δ^5′,8′-3,4,3′,5′-tetramethoxy-4′-oxo-8.1′,7.O.6′-neolignan and rel-(7R,8S,1′R,2′S)-Δ^4′,8′-2′-hydroxy-3,4-dimethoxy-3′-oxo-8.1′,7.O.2′-neolignan.     

Requirements for polyadenylation at the 3' end of LINE-1 elements

LINE-1 (L1) is the only active, autonomous, non-LTR, human retroelement. There are about 5 × 10⁵ L1 copies in the human genome, the majority of which are truncated at their 5′ ends. Both truncated and full-length L1 insertions contain a polyadenylation (polyA) signal at their 3′ ends. A typical polyA site consists of the three main cis-acting elements: a conserved hexamer, cleavage site, and a GU-rich downstream region. A newly inserted L1 copy contains the conserved AATAAA hexamer at the end of its sequence. However, the GU-rich downstream region has to be provided by the neighboring genomic sequences and therefore it would vary for every L1 copy. Using northern blot analysis of transiently transfected L1 expression vectors we demonstrate that L1 element contain sequence that allow efficient polyadenylation at the L1 3′ end upon retrotransposition into a new genomic location independent of the base composition downstream of the insertion site. The strategy of polyadenylation at the 3′ end of L1 parallels the approach the element employs at its 5′UTR by having an unusual internal polymerase II promoter, making new insertions less dependent on the properties of the flanking sequences at the new locus.     

Multiple large segment deletion method for Corynebacterium glutamicum

A precise and scarless genome excision method, employing the Cre/loxP system in concert with double-strand break (DSB)-stimulated intramolecular recombination was developed. The DSBs were mediated by the restriction endonuclease, I-SceI. It permitted multiple deletions of independent 14-, 43-, and 10-kb-long genomic regions on the Corynebacterium glutamicum genome. Accuracy of deletion was confirmed by the loss of marker genes, PCR, and sequencing of new genome joints. Eleven, 58, and 4 genes were predicted on the 14-, 43-, and 10-kb deleted regions, respectively. Although the resultant mutant lost a total of 67 kb encoding 73 genes, it still exhibited normal growth under standard laboratory conditions. Such a large segment deletion method in which multiple, successive deletions are possible is useful for genome engineering.     

Further farnesyl-homogentisic acid derivatives from Otoba parvifolia

The seeds of Otoba parvifolia contain three novel compounds apparently derived from homogentisic acid, rel-(1′R,5′R)-2-(1′-farnesyl-5′-hydroxy-2′-oxocyclohex-3′-en-1′-yl)-acetic acid and its acetate as well as rel-(1′R,4′S,5′R)-2-(1′-farnesyl-4′,5′-dihydroxy-2′-oxocyclohexan-1′-yl)-acetic acid δ-lactone. The structure of an additional isolate, previously described as 2-(1′-farnesyl-2′-hydroxy-5′-oxocyclohex-3′-en-1′-yl)-acetic acid γ-lactone was revised to rel-(1′R,5′R)-2-(1′-farnesyl-5′-hydroxy-2′-oxocyclohex-3′-en-1′-yl)-acetic acid δ-lactone.     

Genomic organization of four β-1,4-endoglucanase genes in plant-parasitic cyst nematodes and its evolutionary implications

The genomic organization of genes encoding β-1,4-endoglucanases (cellulases) from the plant-parasitic cyst nematodes Heterodera glycines and Globodera rostochiensis (HG-eng1, Hg-eng2, GR-eng1, and GR-eng2) was investigated. HG-eng1 and GR-eng1 both contained eight introns and structural domains of 2151 and 2492 bp, respectively. HG-eng2 and GR-eng2 both contained seven introns and structural domains of 2324 and 2388 bp, respectively. No significant similarity in intron sequence or size was observed between HG-eng1 and HG-eng2, whereas the opposite was true between GR-eng1 and GR-eng2. Intron positions among all four cyst nematode cellulase genes were conserved identically in relation to the predicted amino acid sequence. HG-eng1, GR-eng1, and GR-eng2 had several introns demarcated by 5′-GC…AG-3′ in the splice sites, and all four nematode cellulase genes had the polyadenylation and cleavage signal sequence 5′-GAUAAA-3′—both rare occurences in eukaryotic genes. The 5′- flanking regions of each nematode cellulase gene, however, had signature sequences typical of eukaryotic promoter regions, including a TATA box, bHLH-type binding sites, and putative silencer, repressor, and enhancer elements. Database searches and subsequent phylogenetic comparison of the catalytic domain of the nematode cellulases placed the nematode genes in one group, with Family 5, subfamily 2, glycosyl hydrolases from Scotobacteria and Bacilliaceae as the most homologous groups. The overall amino acid sequence identity among the four nematode cellulases was from 71 to 83%, and the amino acid sequence identity to bacterial Family 5 cellulases ranged from 33 to 44%. The eukaryotic organization of the four cyst nematode cellulases suggests that they share a common ancestor, and their strong homology to prokaryotic glycosyl hydrolases may be indicative of an ancient horizontal gene transfer.     

SV40 recombinants carrying a d(CT.GA)22 sequence show increased genomic instability.

Repetitive d(CT.GA)n sequences are commonly found in eukaryotic genomic DNA. They are frequently located in sites involved in genetic recombination or in promoter regions. To test for their possible biological function, a d(CT.GA)22 synthetic sequence was introduced into the genome of SV40, since it constitutes an appropriate model system for eukaryotic chromatin. When SV40 infects permissive cells, it proliferates in the form of a minichromosome. The simple repetitive sequence indicated above was inserted at the unique HpaII site of SV40 (at nt 346), and the genomic stability of SV40 recombinants carrying the d(CT.GA)22 sequence (SV/CT22 viruses) was analyzed. Upon serial passage through permissive CV1 cells, SV/CT22 recombinants show an increased production of defective viruses. Generation of SV/CT22 variants is likely to take place via recombination between and within viral molecules. The enhancement of the rate of recombination induced by the repetitive sequence is likely to be related to its known propensity to form triple-stranded structures. Many different variants coexist in the same viral population indicating that the mechanism by which they are produced is not unique. One variant (SV/X), showing a replicative advantage, was characterized in detail. Variant SV/X accounts for a large proportion of the total viral population. Its genomic organization corresponds to a tandem duplication of an early SV40 DNA fragment spanning from approx. nt 3200-nt 160. Variant SV/X contains a duplicated SV40 ori.     

Cre-stimulated recombination at loxP-containing DNA sequences placed into the mammalian genome.

The cre gene of coliphage P1 encodes a 38 kDa protein which efficiently promotes both intra- and intermolecular recombination at specific 34 bp sites called loxP. To demonstrate that the Cre protein can promote DNA recombination at loxP sites resident on a mammalian chromosome, a mouse cell line was constructed containing two directly repeated loxP sites flanking a 2.5 kb yeast DNA fragment and inserted between the SV40 promoter and the neo structural gene to disrupt expression of the neo gene. Expression of the cre gene in this cell line results in excision of the intervening yeast DNA and thus permits sufficient expression of the neo gene to allow cell growth in high concentrations of G418. Southern analysis indicated that Cre-mediated excision occurred at the loxP sites. In the absence of the cre gene such excisive events are quite rare. Cre-mediated recombination should thus be quite useful in effecting a variety of genomic rearrangements in eukaryotic cells.