Frequency and genetic background of the position 122 (Val----Ile) variant transthyretin gene in the black population.

Transthyretin (TTR) (122 Val----Ile), caused by a point mutation which destroys a MaeIII restriction site, is associated with cardiac amyloidosis in black individuals. To estimate the frequency of the MaeIII(-) gene in the black population without overt cardiac disease, DNA from 177 black individuals without amyloidosis was amplified by the PCR around TTR codon 122 and was digested with MaeIII. The MaeIII(-) gene frequency was 4/354 (1.1%; 95% confidence interval 0.32%2.7%), suggesting that the variant is relatively common in blacks. HLA genotype testing did not suggest that the TTR (122 Val----Ile) heterozygotes were of a closely related genetic background.     

Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific.

The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream.     

Phenylketonuria in U.S. blacks: molecular analysis of the phenylalanine hydroxylase gene

We investigated the frequency, origin, and molecular basis of phenylketonuria (PKU) in U.S. blacks. On the basis of 10 years of Maryland newborn-screening data, we found the frequency to be 1/50,000, or one-third that in whites. We performed haplotype analysis of the phenylalanine hydroxylase (PAH) gene of 36 U.S. blacks, 16 from individuals with classical PKU and 20 from controls. In blacks, 20% of wild-type PAH alleles have a common Caucasian haplotype (i.e., haplotype 1), whereas 80% had a variety of haplotypes, all rare in Caucasians and Asians. One of these, haplotype 15, accounted for a large fraction (30%). Among black mutant PAH alleles, 20% have a haplotype (i.e., either haplotype 1 or haplotype 4) common in Caucasians; 40% have a haplotype rare in Caucasians and Asians, and 40% have one of two previously undescribed haplotypes. Both can be derived from known haplotypes by a single event. One of these haplotypes is characterized by a new MspI restriction site, located in intron 8, which was present in five of 16 black mutant alleles but was not present in 60 U.S. black control, 20 U.S. Caucasian control, or 20 Caucasian mutant PAH alleles. Sequence analysis of DNA from a single individual, homozygous for the new MspI associated haplotype, shows homozygosity for a C----T transition at nucleotide 896 in exon 7 of the PAH cDNA, resulting in the conversion of leucine 255 to serine (L255S).     

Identification of Hb Hamilton or beta 11(A8)Val----Ile gene by the polymerase chain reaction amplification technique.

Amplification of the beta-globin gene by the PCR technique, followed by the enzymatic digestion of the DNA fragment obtained, was used to easily identify the human beta-globin variant Hb Hamilton which is characterized by the valine to isoleucine substitution at position 11. The result revealed the predicted G to A transition at codon 11 which abolishes a MaeIII restriction site. This mutation, which is rather common among Sardinians, is at the level of one of the five CpG dinucleotides of the beta-globin gene.     

Phylogenetic analysis and confirmation of the endospore-forming nature of Pasteuria penetrans based on the spo0A gene

Pasteuria penetrans is an obligate parasite of plant parasitic nematodes and has yet to be grown in vitro. We have cloned the pivotal sporulation gene, spo0A, which is the first whole gene yet to come from this organism. Partial spo0A sequences were also obtained from the related bacteria, Pasteuria ramosa and Alicyclobacillus acidocaldarius. Phylogenetic analyses using the spo0A sequence data from this and previous studies confirmed the closeness of the genera Pasteuria and members of the supergenus Bacillus. A segment of the spo0A gene was also used to show that genetic heterogeneity exists within and between populations of P. penetrans. This may explain, partly at least, the variability of P. penetrans as a biological control agent of nematodes.     

Evidence supporting a single origin of the beta(C)-globin gene in blacks.

In order to characterize the origin(s) of the beta C-globin gene in blacks, 25 chromosomes bearing this gene were characterized at eight polymorphic restriction sites within the beta-globin gene cluster. Twenty-two of the 25 chromosomes were identical at all sites and possessed a haplotype seen only infrequently among beta A-bearing chromosomes in black Americans. Two different haplotypes were observed among the three exceptional chromosomes. These haplotypes were identical to the most common beta C allele in the 3' end of the beta-globin gene cluster, but differed in the 5' region. Partial haplotype analysis on an additional 14 beta C alleles demonstrated complete association with the typical beta C-associated polymorphisms in the 3' region of the cluster. These data can be most easily explained by a single origin of the mutation followed by spread of the mutation to other haplotypes through meiotic recombination 5' to the beta-globin gene.     

A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase

Polymerase chain reaction (PCR) primers that flank a 688-bp segment within the glutamate dehydrogenase gene (gdh) of Streptococcus suis type 2 could amplify efficiently the DNA of all 306 (100%) clinical S. suis isolates tested (pigs, n=305; human, n=1) encompassing all serotypes obtained from diverse organs, and geographic origins. When DNA from other bacteria were used as templates for amplification, no product was detected indicating specificity of the primers. Multiplex PCR was developed using the gdh gene primer pair and primers that targeted the gene encoding S. suis capsular biosynthesis (cps). This strategy enabled the detection of strains belonging to serotypes 1/2, 1, 2, 7, and 9, respectively. Using the multiplex-PCR technique, 12 out of 14 (86%) isolates that were previously identified as non-typable S. suis (based on biochemical reactions and serology) gave positive PCR results of which four were positive for serotype 7, three for serotype 2, and five for S. suis strains that belong to other serotypes. Retest results of all 14 isolates by several veterinary laboratories were identical with PCR and confirmed that the two non-PCR reactive isolates belonged to strains of other streptococcal species. These results indicated that PCR improved species determination and can thus be used as a reliable species-specific molecular diagnostic reagent for the accurate identification of S. suis isolates and a serotype-specific method for the detection of strains of serotypes 1/2, 1, 2, 7, and 9, respectively. The PCR method therefore has potential clinical and epidemiological applications.     

Endogenous gibberellins in immature seeds of Prunus persica L.: identification of GA(118), GA(119), GA(120), GA(121), GA(122) and GA(126)

The endogenous gibberellins in immature seeds of Prunus persica were analyzed by gas chromatography-mass spectrometry. Eleven known gibberellins, GA(3), GA(9), GA(17), GA(19), GA(30), GA(44), GA(61), GA(63), GA(87), GA(95) and GA(97) were identified. Additionally, several hitherto unknown gibberellins were detected and their putative structures were verified by synthesis of the authentic gibberellins. These gibberellins were then assigned trivial numbers, e.g. 1alpha-hydroxy GA(20) (GA(118)), 1alpha-hydroxy GA(9) (GA(119)), 1,2-didehydro GA(9) (GA(120)), 1,2-didehydro GA(70) (GA(121)), 1,2-didehydro GA(69) (GA(122)) and 1,2-didehydro GA(77) (GA(126)). GA(118) and GA(119) were the first 1alpha-hydroxy gibberellins identified from higher plants. The above profile of 1,2-didehydro gibberellins suggests that 1,2-dehydrogenation might occur prior to 3beta-hydroxylation in biosynthesis of GA(3), GA(30) and GA(87) in immature seeds of P. persica.     

A rapid sex-identification test for the forest musk deer (Moschus berezovskii) based on the ZFX/ZFY gene

We describe a rapid sex-identification method for the forest musk deer (Moschus berezovskii) using PCR based on zinc-finger protein-encoding genes (ZFX/ZFY) located on the X and Y chromosomes. Fragments of the ZFX and ZFY genes were amplified and sequenced. The ZFX and ZFY fragments were identical in length and 94% similar in nucleotide sequence. Specific primers for forest musk deer sex identification were designed on the basis of sequence differences between ZFX and ZFY. All the primers were multiplexed in single-tube PCR. Both male and female forest musk deer showed amplification bands of 447 bp and 212 bp separated in agarose gels. A sex-specific 278-bp band was amplified only from males. These results show that testing by PCR for the presence of the 278-bp sequence is a rapid and reliable method for sex identification.     

Phylogenetic analysis of Helianthus (Asteraceae) based on chloroplast DNA restriction site data

 Chloroplast DNA (cpDNA) restriction site data were used to analyze phylogenetic relationships within Helianthus. Data from new samples were consistent with previous results and showed the genus to be a well-defined (11 site changes) lineage within subtribe Helianthinae. The affinities of 2 species, Viguiera similis and V. phenax (V. ludens) that have sometimes been included in Helianthus were shown to lie outside the genus. The two species of Phoebanthus, however, formed a phylogenetic ingroup to the perennial Helianthus. Within the genus, cpDNA data indicated that there are four distinct phylogenetic lineages. Two of the lineages each contained a single, annual species (H. agrestis and H. porteri, respectively). The remaining annual species collectively formed a third lineage (sect. Helianthus). The fourth lineage contained all of the perennial species, including those of Phoebanthus. Within the perennial lineage, there was little variation in restriction site characters. The 3 species of series Pumili formed a paraphyletic group at the base of the perennial lineage, and the 3 species of series Ciliares formed a group that was supported by a single restriction site character. Received: 2 September 1996 / Accepted: 25 October 1996     

Evidence from intron 1 of the nuclear transthyretin (Prealbumin) gene for the phylogeny of African mole-rats (Bathyergidae)

A 900- to 1100-bp fragment encompassing intron 1 of the nuclear transthyretin (prealbumin) gene was examined in 12 taxa of Old World hystricognath rodents of the families Bathyergidae, Petromuridae, Thryonomyidae, and Hystricidae. Within the Bathyergidae, Heterocephalus glaber (naked mole-rat) was basal, and the other East African species, Heliophobius argenteocinereus (silvery mole-rat), was sister to a southern African clade containing Bathyergus, Cryptomys, and Georychus (dune, common, and cape mole-rats). These results are congruent with studies using mitochondrial 12S rRNA gene sequences. A combined analysis of transthyretin and 12S rRNA data resulted in a well-supported topology with better resolution than either gene analyzed separately. These data support the findings by M. W. Allard and R. L. Honeycutt (1992, Mol. Biol. Evol. 9: 27-40) and R. L. Honeycutt (1992, Am. Sci. 80: 43-53) that complex social systems evolved independently at least twice, in the common and naked mole-rats.     

A specific DNA sequence is required for high frequency of recombination in the ade6 gene of fission yeast.

The point mutation M26 in the ade6 gene of Schizosaccharomyces pombe increases recombination frequency by an order of magnitude in comparison with other mutations in the same gene. The hypothesis is tested that this hot spot of recombination requires a specific nucleotide sequence at the M26 site. The DNA sequence is altered systematically by in vitro mutagenesis, and the resulting sequences are introduced into the ade6 gene in vivo by gene replacement. It results that any change of the heptanucleotide ATGACGT leads to loss of high frequency of recombination. Thus this oligonucleotide sequence is necessary for high frequency of recombination, but it seems not to be sufficient.     

Assignment of T cell receptor (TCR) alpha-chain gene (A), beta-chain gene (B), gamma-chain gene (G), and delta-chain gene (D) loci on swine chromosomes by in situ hybridization and radiation hybrid mapping

Using the cDNA sequence of porcine T-cell receptor (TCR) alpha-, beta-, gamma-, and delta-chain genes, we screened a porcine BAC library to isolate clones containing these genes. The isolated BAC clones were confirmed to carry these TCR genes by partial nucleotide sequencing. Among the clones obtained in the present screening, two clones carried both the TCR alpha-chain gene (TRA) and the TCR delta-chain gene (TRD) while one clone each carried only the sequence of either TRA or TRD. This observation demonstrated that TRA and TRD are localized in close proximity on a swine chromosome. Also two clones contained the sequence of the TCR beta-chain gene (TRB), and two clones contained the sequence of TCR gamma-chain gene (TRG). Fluorescence in situ hybridization using the above BAC clones as probes revealed that TRA and TRD, TRB, and TRG loci reside on swine chromosomes 7q15.3-->q21, 18q11.3-->q12, and 9q21-->q22, respectively. The chromosome positions of TRA and TRB are consistent with those determined by somatic cell hybrid analysis (Rettenberger et al., 1996). In addition, RH mapping of these genes was performed using the INRA-University of Minnesota RH panel DNA. The result confirmed the position of TRA and TRB reported earlier (http://imprh.toulouse.inra.fr/), and further demonstrated that TRG was located 11 cR away from genetic marker SW989 toward the marker S0019.     

A molecular phylogeny of the Cephinae (Hymenoptera, Cephidae) based on mtDNA COI gene: a test of traditional classification

Cephinae is traditionally divided into three tribes and about 24 genera based on morphology and host utilization. There has been no study testing the monophyly of taxa under a strict phylogenetic criterion. A molecular phylogeny of Cephinae based on a total of 68 sequences of mtDNA COI gene, representing seven genera of Cephinae, is reconstructed to test the traditional limits and relationships of taxa. Monophyly of the traditional tribes is not supported. Monophyly of the genera are largely supported except for Pachycephus. A few host shift events are suggested based on phylogenetic relationships among taxa. These results indicate that a more robust phylogeny is required for a more plausible conclusion. We also report two species of Cephus for the first time from Turkey.     

DNA restriction and modification in Escherichia coli: functional analysis of the role of the dnaC(D) gene product

Escherichia coli strain PC-7 carries two independent temperature-sensitive mutations, one affecting the restriction and modification (R-M) phenotype and the other the DnaC(D) phenotype. The results of complementation and P1 transduction analysis of the mutation affecting the R-M phenotype implicate a fourth gene, designated hsdX, located close to the hsd three-gene complex. The properties of merodiploids constructed between appropriate recipients and F' elements with different mutations in hsdS, hsdR and hsdM genes might indicate that in strain PC-7 the temperature-sensitive products, determined by hsdR and hsdSK cistrons, are synthesized. The role of the temperature-sensitive dnaC(D) gene product in the formation of the restriction endonuclease was studied and no direct relation was found between the DnaC(D) and R-M phenotypes.     

A new method for analyzing gene expression based on frequency analysis of RT-PCR products obtained with degenerate primers

This paper presents a new method of gene expression analysis: frequency analysis of RT-PCR products obtained with degenerate primers (FAPP). The main advantage of the new approach compared to the present methods of gene expression analysis is that it is applicable to non-model plant objects whose gene sequences are unknown. The advantages and disadvantages of FAPP are described in detail using data on calcium-dependent protein kinase, stilbene synthase, and phenylalanine ammonia-lyase gene expression in two cell cultures of Vitis amurensis. We compared the expression profiles obtained by FAPP to those obtained by real-time PCR and expressed sequence tags.     

A phylogenetic analysis of Doronicum (Asteraceae, Senecioneae) based on morphological, nuclear ribosomal (ITS), and chloroplast (trnL-F) evidence.

A phylogenetic analysis of the Old World genus Doronicum (26 species, 4 subspecies) based on sequence data of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA, the chloroplast spacer trnL-F, and morphology is presented. Congruence among the three data sets was explored by the computing of several indices, all of which suggest homogeneity between only the two molecular matrices. We argue that the morphological data set contains poor phylogenetic signal and advocate simultaneous analysis of the three data sets (total evidence approach) so that morphological characters are tested for homology by congruence with molecular data. The resulting phylogenetic hypothesis allows several well-supported conclusions including the placement of a Corsican endemic (D. corsicum), sister to the remainder of the genus, and the inference that an early southern European or Mediterranean diversification took place in the genus. Shifts in morphological characters (e.g., homocarpy to heterocarpy) are confirmed to have evolved several times. Results from comparative studies of sequence data of the chloroplast gene ndhF support inclusion of Doronicum in tribe Senecioneae.