Genomic fingerprints produced by PCR with consensus tRNA gene primers.

The polymerase chain reaction using only a single 'consensus' tRNA gene primer, or a pair of primers facing outward from tRNA genes, amplifies a set of DNA fragments in bacterial, plant and animal genomic DNAs. Presumably, these PCR fingerprints are mainly derived from the regions between closely linked tRNA genes. The pattern of the PCR products is determined by which genomes and which primer(s) are used. Genomic fingerprints are largely conserved within a species and, in bacteria, most products in the fingerprint are conserved between closely related species. Thus, PCR with tRNA gene consensus primers helps to identify species and genera.     

Targeting DNA with triplex-forming oligonucleotides to modify gene sequence

Molecules that interact with DNA in a sequence-specific manner are attractive tools for manipulating gene sequence and expression. For example, triplex-forming oligonucleotides (TFOs), which bind to oligopyrimidine.oligopurine sequences via Hoogsteen hydrogen bonds, have been used to inhibit gene expression at the DNA level as well as to induce targeted mutagenesis in model systems. Recent advances in using oligonucleotides and analogs to target DNA in a sequence-specific manner will be discussed. In particular, chemical modification of TFOs has been used to improve binding to chromosomal target sequences in living cells. Various oligonucleotide analogs have also been found to expand the range of sequences amenable to manipulation, including so-called "Zorro" locked nucleic acids (LNAs) and pseudo-complementary peptide nucleic acids (pcPNAs). Finally, we will examine the potential of TFOs for directing targeted gene sequence modification and propose that synthetic nucleases, based on conjugation of sequence-specific DNA ligands to DNA damaging molecules, are a promising alternative to protein-based endonucleases for targeted gene sequence modification.     

Nucleotide sequence and expression in E. coli of a human interferon-alpha gene selected from a genomic library using synthetic oligonucleotides

The complete nucleotide sequence of a human interferon-alpha gene is reported. The gene, designated IFN-alpha M1, was isolated from a human genomic library in phage lambda Charon 4A using synthetic oligonucleotides as hybridization probes. Based on a comparison of nucleotide sequence data obtained from this recombinant phage with published interferon-alpha gene sequences, a region of DNA capable of coding for a pre-interferon of 189 amino acids was identified. An AluI fragment containing the coding region for the mature interferon was inserted into the HincII site of the phage M13mp11, resulting in a fusion of portions of the IFN-alpha M1 and the beta-galactosidase genes. Antiviral activity was detected in extracts from E. coli infected with the recombinant M13 phage carrying the fused gene. The antiviral activity was completely neutralized by antibodies to human interferon-alpha.     

Genome sequence of Aggregatibacter actinomycetemcomitans RHAA1, isolated from a rhesus macaque, an Old World primate

Aggregatibacter actinomycetemcomitans is implicated in localized aggressive periodontitis. We report the first genome sequence of an A. actinomycetemcomitans strain isolated from an Old World primate.     

Bifidobacterium primatium sp. nov., Bifidobacterium scaligerum sp. nov., Bifidobacterium felsineum sp. nov. and Bifidobacterium simiarum sp. nov.: Four novel taxa isolated from the faeces of the cotton top tamarin (Saguinus oedipus) and the emperor tamarin (Saguinus imperator)

Four novel Gram-stain-positive, non spore forming and fructose-6-phosphate phosphoketolase-positive strains were isolated from the faeces of a cotton top tamarin (Saguinus oedipus) and an emperor tamarin (Saguinus imperator). Phylogenetic analyses based on 16S rRNA revealed that bifidobacterial strains TRE 1^T exhibit close phylogenetic relatedness to Bifidobacterium catulorum DSM 103154 (96.0%) and Bifidobacterium tissieri DSM 100201 (96.0%); TRE D^T and TRE H^T were closely related to Bifidobacterium longum subsp. longum ATCC 15708^T with similarity values of 97.4% and 97.5%, respectively; TRI 7^T was closely related to Bifidobacterium tissieri DSM 100201 (96.0%). The Average Nucleotide Identity (ANI) and in silico DDH (isDDH) analysis with closest neighbour supported an independent phylogenetic position of all strains with values ranged from 74 to 85% for ANI and from 24 to 28% for isDDH. DNA base composition of the four strains was in the range of 58.3–63.5 mol% G + C. Based on the phylogenetic, genotypic and phenotypic data, the strains TRE 1^T, TRE D^T, TRE H^T and TRI 7^T clearly represent four novel taxa within the genus Bifidobacterium for which the names Bifidobacterium primatium sp. nov. (type strain TRE 1^T = DSM 100687^T = JCM 30945^T), Bifidobacterium scaligerum sp. nov. (type strain TRE D^T = DSM 103140^T = JCM 31792^T), Bifidobacterium felsineum sp. nov. (type strain TRE H^T = DSM 103139^T = JCM 31789^T) and Bifidobacterium simiarum sp. nov. (type strain TRI 7^T = DSM 103153^T = JCM 31793) are proposed.     

1H-NMR conformational study of a synthetic peptide derived from the consensus sequence of annexins.

The solution conformation of a synthetic 18 amino acid peptide derived from a consensus sequence of Annexins has been investigated by 1H NMR. Full sequential assignment has been achieved. Conformational properties of the peptide were deduced from the analysis of J(NH-CH alpha) coupling constants, amide proton exchange, 2D NOESY connectivities and computer modeling.     

Roles of IFN consensus sequence binding protein and PU.1 in regulating IL-18 gene expression.

IL-18 is expressed from a variety of cell types. Two promoters located upstream of exon 1 (5'-flanking region) and upstream of exon 2 (intron 1) regulate its expression. Both promoter regions were cloned into pCAT-Basic plasmid to yield p1-2686 for the 5'-flanking promoter and p2-2.3 for the intron 1 promoter. Both promoters showed basal constitutive activity and LPS inducibility when transfected into RAW 264.7 macrophages. To learn the regulatory elements of both promoters, 5'-serial deletion and site-directed mutants were prepared. For the activity of the p1-2686 promoter, the IFN consensus sequence binding protein (ICSBP) binding site between -39 and -22 was critical. EMSA using an oligonucleotide probe encompassing the ICSBP binding site showed that LPS treatment increased the formation of DNA binding complex. In addition, when supershift assays were performed, retardation of the protein-DNA complex was seen after the addition of anti-ICSBP Ab. For the activity of the p2-2.3 promoter, the PU.1 binding site between -31 and -13 was important. EMSA using a PU.1-specific oligonucleotide demonstrated that LPS treatment increased PU.1 binding activity. The addition of PU.1-specific Ab to LPS-treated nuclear extracts resulted in the formation of a supershifted complex. Furthermore, cotransfection of ICSBP or PU.1 expression vector increased p1 promoter activity or IL-18 expression, respectively. Taken together, these results indicate that ICSBP and PU.1 are critical elements for IL-18 gene expression.     

Reproduction of the emperor tamarin (Saguinus imperator) in captivity,with comparisons to cotton-top and golden lion tamarins

Little information has been published on the reproductive biology and behavior of the emperor tamarin (Saguinus imperator). We analyzed twelve years of data on emperor tamarins at the Los Angeles Zoo and made comparisons with data on cotton-top tamarins (Saguinus o. oedipus) and golden lion tamarins (Leontopithecus rosalia) from the same collection. Secondary sex ratios did not differ significantly from 50:50. Births were not strictly seasonal for any species. The number of infants reared had a significant effect on interbirth interval for all species, with shorter intervals when only one or no infants were reared, but females did sometimes conceive early in lactation. In emperor tamarin families, all fathers and most older siblings carried new infants, usually beginning within a few days after a birth. Previous exposure to younger siblings did not appear to be critical to the development of competent parental behavior by zooborn emperor tamarin females.     

Nucleotide sequence of the amicyanin gene from Methylobacterium extorquens AM1.

The gene for amicyanin from the methylotrophic bacterium, Methylobacterium extorquens AM1 was identified. It encodes a protein consisting of 119 amino acids with a molecular weight of 12,609 kDa. The amino acid sequence shows the presence of a typical leader sequence and signal peptidase recognition site. Two putative hairpin structures were found, one located directly behind the amicyanin gene and another located 50 bp upstream. The same sequence AAAATCCC was found near the start codons for the small subunit of methylamine dehydrogenase and amicyanin, but its significance is not known.     

Alteration of the LIS1 gene in Japanese patients with isolated lissencephaly sequence or Miller-Dieker syndrome

LIS1 is a genetic entity that is responsible for lissencephaly. Previously we have reported isolated lissencephaly sequence(ILS) in a Japanese patient carrying a balanced chromosomal translocation that disrupted the LIS1 gene. We examined mutations of LIS1 in 12 additional Japanese patients, 8 of them with ILS and 4 with Miller-Dieker syndrome (MDS). Fluorescence in situ hybridization (FISH) analysis disclosed deletions of part of the LIS1 gene or of the chromosomal region surrounding it in three of the ILS cases and in three of the MDS cases. In one of the remaining five ILS cases, SSCP analysis and subsequent sequence analysis identified a 1-bp deletion in exon IV, which can be expected to result in premature termination of the gene product. Our results indicate that in Japan, as elsewhere, abnormality of the LIS1 gene is a common cause of MDS/ILS. Received: 20 April 1998 / Accepted: 28 July 1998     

Molecular sex identification of tinamous with PCR using primers derived from the spindlin gene

This paper describes results which show that the spindlin gene has different forms on the tinamid W and Z chromosome, providing a sensitive and accurate procedure for a molecular, polymerase chain reaction‐based procedure for sex identification of tinamous.     

Single locus microsatellites isolated using 5' anchored PCR.

Microsatellites are widely used as genetic markers because they are co-dominant, multiallelic, easily scored and highly polymorphic. A major drawback of microsatellite markers is the time and cost required to characterise them. We have developed a novel technique to reduce this cost by producing a microsatellite-rich PCR profile from genomic DNA which was cloned to yield a genomic library enriched for microsatellites. Sequence data and subsequent allele scoring within pedigrees revealed that these microsatellites retained their original repeat length and segregated normally. This technique permits genomic amplification with only one specific primer. Together with enrichment, the savings in primer costs reduces the cost of microsatellite characterisation considerably.