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PubMed1991

Distribution of the salmonid Hpa 1 family in the salmonid species demonstrated by in vitro runoff transcription assay of total genomic DNA: a procedure to estimate repetitive frequency and sequence divergence of a certain repetitive family with a few known sequences.

An in vitro runoff transcription assay of total genomic DNA was developed. As an example of use of this assay, analysis of a highly repetitive sequence in the cherry salmon (Oncorhynchus masou) is described. Total genomic DNA of the cherry salmon was completely digested with Hpa 1, whose site is known to be in the tRNA-unrelated region of the cherry salmon Hpa 1 family. On transcription of the digested DNA in a HeLa cell extract, a discrete-sized RNA of about 100 nucleotides, constituting 70% of the transcripts, was produced, whereas on transcription of the undigested total DNA, only smeared RNA was obtained. In a fingerprint, the oligonucleotides of the discrete transcript from the digested total DNA were very distinct and exactly corresponded to those of a transcript from an Hpa 1 digest of a cloned DNA, but with few extra oligonucleotides. These results showed that the cherry salmon Hpa 1 family constitutes a major repetitive family in the genome of the cherry salmon. For determination of the distribution of the salmonid Hpa 1 family in other salmonid species, the same analysis was applied to DNAs from the chum salmon (Onchorhynchus keta), brown trout (Salmo trutta), Japanese common charr (Salvelinus leucomaenis pluvius), and Japanese huchen (Hucho perryi). The results showed that the salmonid Hpa 1 family is widespread in the genomes of salmonid species. A method and equations are also presented for estimating the relationship between the ratio of a given repetitive family to all the Pol III genes and its average sequence divergence by calculating the molar ratio of the runoff transcript to all the in vitro Pol III transcripts.

Animals↗
PubMed1996

DNA microsatellites in domesticated dogs: application in paternity disputes.

Microsatellite sequences, like minisatellites, are a class of polymorphic DNA fragments that are randomly distributed throughout mammalian genome. Although they express significantly lower variation than minisatellites, they have potential to be used in paternity disputes. However, the inherently lower variability together with the more genetically homogeneous nature of pedigree dogs due to the major inbreeding, raised doubts about the effectiveness of microsatellites in canine paternity (paternage) testing. Using about 20 polymorphic canine specific microsatellites on various dog litters demonstrated, that canine microsatellites provide a perfectly adequate basis for assigning paternity in pedigree breeds. Microsatellite paternity tests are more straightforward to perform and interpret than those based on minisatellites (DNA fingerprinting) and require as little as 0.1 ml of blood. The case presented here was a paternity dispute in labradors, where a mixed paternity was suspected.

Alleles↗
PubMed1993

The origin and genetic differentiation of native breeds of pigs in southwest China: an approach from mitochondrial DNA polymorphism.

Mitochondrial DNA (mtDNA) of six breeds of native domestic pigs from Yunnan province, southwest China, and two wild boars obtained from Sichuan, China, and Vietnam was analyzed using 20 restriction endonucleases that recognize six nucleotides. Restriction maps were made by double-digestion methods and polymorphic sites were located on the map. According to their mtDNA restriction types, all the breeds were classified into six groups. Genetic distances among groups were calculated to define their phylogenetic relationships. The relationship between the Sichuan wild boar and domestic pigs is close, while the Vietnamese wild boar is relatively far from them, so the domestic pigs in southwest China are likely to have originated from a wild pig which distributed in west China. We compare our results with previous reports in literature and discuss the relationship among Chinese pigs, Japanese pigs, and European pigs. The mtDNA cleavage pattern of the Mingguang pig digested by EcoRV was identical to that of Duroc; mutations at the EcoRI site, detected in the mtDNA of two Dahe pigs, are the same as in the Vietnamese wild boar, suggesting that mutational hot spots exist in the mtDNA of pigs.

Animals↗
PubMed2005

Genes and homology in nervous system evolution: comparing gene functions, expression patterns, and cell type molecular fingerprints.

The evolution of the nervous system is one of the most fascinating, but also most nebulous fields of homology research. We do not know for example whether the last common ancestors of human, squid, and fly already possessed an elaborate brain and eyes, or rather had a simple, diffuse nervous system. Nevertheless, in the past decade molecular data has greatly advanced our understanding of bilaterian nervous system evolution. In this methodological review, I explain the four levels on which molecular genetic studies advance the quest for homologies between animal nervous systems. (I) Bioinformatic homology research elucidates the evolutionary history of gene families relevant for nervous system evolution such as the opsin superfamily. It tells us when and in what order genes and their functions have emerged. Based on this, we can (II) infer the organismal complexity of some remote ancestor from the functional diversity of its reconstructed proteome. (III) Most common in molecular homology research has been the comparison of expression patterns of developmental control genes. This approach matches and aligns embryonic regions along the body axes, between remote bilaterians. It does not tell us much, however, about the complexity of structures that developed from these regions in Urbilateria. (IV) This is overcome by a novel variant of molecular homology research, the comparison of cell types. Here, a similar "molecular fingerprint" of cells is taken as indication of cross-bilaterian homology. This approach makes it possible to reconstruct the cell-type repertoire of the urbilaterian nervous system.

Animals↗
PubMed1998

The gene for lysosomal protein CD63 is normal in patients with Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) is one of the few genetic disorders associated with severe pulmonary fibrosis. Fifty percent of affected patients die as a result of respiratory insufficiency. Fibrosis is thought to be caused by the accumulation of ceroid, an insoluble fluorescent lipoprotein, both extracellularly and in the lysosomes of alveolar macrophages. In addition to pulmonary fibrosis, HPS is characterized by oculocutaneous albinism and a reduction in the number of platelet dense bodies. CD63 is a protein that was described originally in platelet lysosomes. It localizes to the membranes of melanosomes and platelet dense bodies. CD63 is decreased dramatically in the lysosomes and dense bodies of patients with HPS. We theorized that CD63, a membrane protein common to lysosomes, melanosomes, and platelet dense bodies, may play a role in HPS. We sought to characterize the gene coding for this protein in HPS lymphoid cell lines. The coding region for CD63 was sequenced in control and HPS cell lines. Messenger RNA from HPS and normal cell lines was examined by Northern analysis. Genomic DNA from the same cell lines was examined by Southern analysis and polymerase chain reaction (PCR). CD63 protein in lymphoid cell lines and peripheral blood monocytes was compared by Western analysis. We found no mutations in the coding region of CD63 in an HPS cell line. We also found no diminution in the quantity of CD63 RNA by Northern analysis and no gross defects in the structural gene by PCR and Southern analysis, suggesting that the CD63 structural gene, promoter, and untranslated regions were normal. Western analysis showed that the 43-kDa protein was present in control and HPS lymphoid cell lines and peripheral blood monocytes in equivalent amounts. Although CD63 is an attractive candidate for the primary defect of HPS, the disease is probably not caused by a mutation in the CD63 gene.

Adult↗
PubMed2004

Use of Pi5(t) markers in marker-assisted selection to screen for cultivars with resistance to Magnaporthe grisea.

Identification of the PCR markers tightly linked to genes that encode important agronomic traits is useful for marker-assisted selection (MAS). The rice Pi5(t) locus confers broad-spectrum resistance to Magnaporthe grisea, the causal agent of rice blast disease. It has been hypothesized that the Pi5(t) locus carries the same gene as that encoded by the Pi3(t) and Pii(t) loci. We developed three PCR-based dominant markers (JJ80-T3, JJ81-T3, and JJ113-T3) from three previously identified BIBAC clones-JJ80, JJ81, and JJ113-that are linked to the Pi5(t) locus. PCR analysis of 24 monogenic lines revealed that these markers are present only in lines that carry Pi5(t), Pi3(t), and Pii(t). PCR and DNA gel-blot analysis of candidate resistance lines using JJ80-T3, JJ81-T3, and JJ113-T3 indicated that Tetep is the likely donor of Pi5(t). Of the 184 rice varieties tested, 34 carried the JJ80-T3-, JJ81-T3-, and JJ113-T3-specific bands. Disease evaluation of those 34 varieties revealed that all conferred resistance to PO6-6. The genomic structure of three of these resistant varieties (i.e., IR72, Taebaeg, Jahyangdo) is most similar to that of Pi5(t). Our results demonstrate the usefulness of the JJ80-T3, JJ81-T3, and JJ113-T3 markers for MAS for M. grisea resistance.

Breeding↗
PubMed2004

A PCR based B-genome-specific marker in Brassica species.

Previous hybridisation studies showed that the repetitive DNA sequence pBNBH35 from Brassica nigra (genome BB, 2n=16) bound specifically to the B-genome and not to the A- or C-genomes of Brassica species. We amplified a sub-fragment of pBNBH35 from B. nigra by PCR, cloned and sequenced this sub-fragment, and confirmed that it was a 329-bp sub-fragment of pBNBH35. PCR and hybridisation techniques were used to confirm that the pBNBH35 sub-fragment was Brassica B-genome-specific. Fluorescence in situ hybridisation (FISH) in B. nigra, B. juncea (AABB, 2n=36) and B. napus (AACC, 2n=38) showed that the pBNBH35 sub-fragment was present on all eight Brassica B-genome chromosomes and absent from the A- and C-genome chromosomes. The pBNBH35 repeat was localised to the centromeric region of each B-genome chromosome. FISH clearly distinguished the B-genome chromosomes from the A-genome chromosomes in the amphidiploid species B. juncea. This is the first known report of a B-genome repetitive marker that is present on all B-genome chromosomes. It will be a useful tool for the detection of B chromosomes in interspecific hybrids and may prove useful for phylogenetic studies in Brassica species.

Base Sequence↗
PubMed2000

An updated and comprehensive rRNA phylogeny of (crown) eukaryotes based on rate-calibrated evolutionary distances.

Recent experience with molecular phylogeny has shown that all molecular markers have strengths and weaknesses. Nonetheless, despite several notable discrepancies with phylogenies obtained from protein data, the merits of the small subunit ribosomal RNA (SSU rRNA) as a molecular phylogenetic marker remain indisputable. Over the last 10 to 15 years a massive SSU rRNA database has been gathered, including more then 3000 complete sequences from eukaryotes. This creates a huge computational challenge, which is exacerbated by phenomena such as extensive rate variation among sites in the molecule. A few years ago, a fast phylogenetic method was developed that takes into account among-site rate variation in the estimation of evolutionary distances. This "substitution rate calibration" (SRC) method not only corrects for a major source of artifacts in phylogeny reconstruction but, because it is based on a distance approach, allows comprehensive trees including thousands of sequences to be constructed in a reasonable amount of time. In this study, a nucleotide variability map and a phylogenetic tree were constructed, using the SRC method, based on all available (January 2000) complete SSU rRNA sequences (2551) for species belonging to the so-called eukaryotic crown. The resulting phylogeny constitutes the most complete description of overall eukaryote diversity and relationships to date. Furthermore, branch lengths estimated with the SRC method better reflect the huge differences in evolutionary rates among and within eukaryotic lineages. The ribosomal RNA tree is compared with a recent protein phylogeny obtained from concatenated actin, alpha-tubulin, beta-tubulin, and elongation factor 1-alpha amino acid sequences. A consensus phylogeny of the eukaryotic crown based on currently available molecular data is discussed, as well as specific problems encountered in analyzing sequences when large differences in substitution rate are present, either between different sequences (rate variation among lineages) or between different positions within the same sequence (among-site rate variation).

Base Sequence↗
PubMed1998

[Diagnostic error in Leber's optic neuropathy. Value of clinical and molecular genetic studies].

BACKGROUND: Leber's hereditary optic neuropathy is associated with point mutations in the mitochondrial DNA (mtDNA) that appear to be pathogenic for this disease. These mutations affect nucleotide positions 3460, 11,778 and 14,484. MATERIALS AND METHODS: We reviewed the clinical and molecular genetic characteristics of 29 visually symptomatic patients with the clinical diagnosis of LHON. RESULTS: Nine patients really suffered from LHON, but in 20 patients other ocular diseases could be proven. Degeneration of the retina and choroid was most common (seven patients), followed by vascular optic disease (six patients). Three patients suffered from tobacco-alcohol amblyopia, two from optic neuritis and two from autosomal dominant optic neuropathy. CONCLUSIONS: The clinical diagnosis of LHON is strengthened by a proven maternal inheritance and clinical signs such as a severe decrease in visual acuity, central or centrocecal scotomas in the perimetry and pseudoedema of the optic disc, followed by optic atrophy. Pathognomonic clinical signs of LHON are twisted vessels and ectatic capillaries in the fundus of these patients and their relatives of the maternal line, i.e., peripapillary microangiopathy. A careful analysis of the patients' pedigrees, anamnesis and the functional and morphological results of the clinical examinations helps to avoid misdiagnosis of the disease. However, the expensive and time-consuming molecular genetic analysis is always necessary to confirm or exclude the diagnosis of LHON.

Adult↗
PubMed1999

[Leber optic neuropathy in women and children].

BACKGROUND: Leber's hereditary optic neuropathy is associated with point mutations in the mitochondrial DNA (mtDNA) that appear to be pathogenetic for this disease. These mutations affect nucleotide positions 3460, 11778 and 14484. Does the clinical course of LHON differ between men, women and children? MATERIALS AND METHODS: We reviewed the clinical and molecular genetic characteristics of 15 visually symptomatic patients with the clinical diagnosis of LHON (11 women and 4 male children) and compared them with 66 men with LHON. RESULTS: LHON was confirmed clinically and with molecular genetic methods in all cases. Men, women and children showed no differences: Classic fundus findings and typical visual field defects were equally found in both sexes. However, age at the beginning of the disease, severity of LHON and rate of spontaneous recovery differed between groups. Women were older (19-55 years, average 31.3 years) than men (15-53 years, average 24.3 years) at the beginning of the disease. Women suffered more severely from LHON. Spontaneous recovery of vision in women was extremely rare. Many more women had a LHON-affected mother than men. All the affected children (9-14, average 11.7 years at the beginning of the disease) did not have a good visual outcome. CONCLUSIONS: There are some differences in the course of LHON between men and women, concerning age, severity of LHON and rate of spontaneous recovery. Children may also have an unfavorable prognosis.

Adolescent↗
PubMed1998

The chromatin structure of the GAL1 promoter forms independently of Reb1p in Saccharomyces cerevisiae.

Positive and negative regulation of the GAL1 promoter of the yeast Saccharomyces cerevisiae results from a network of interactions between transcription factors and chromatin. In this study we used footprinting procedures to characterize these interactions in vivo. DNase I analysis of the GAL1 upstream activating sequence (UAS(GAL1/10)) showed expected Gal4 activator protein binding during growth in galactose, and also revealed binding of the Reb1 protein (Reb1p) during growth in glucose. In addition, we mapped to nucleotide resolution a positioned nucleosome that, in the inactive promoter, packages DNA between the UAS(GAL1/10) and the GAL1 TATA sequence, leaving both of these elements nucleosome free. The nucleosome footprint was lost when the promoter was activated. Surprisingly, mutation of the Reb1p binding site had no effect on nucleosome positioning or on the kinetics or extent of activation or repression of either the GAL1 or GAL10 promoters under any of the conditions assayed.

Base Sequence↗
PubMed1987

RNA fingerprinting using a small horizontal agarose gel electrophoresis apparatus.

The electrophoresis step in the RNA fingerprinting procedure has been modified by replacing the large and expensive electrophoresis tank with a horizontal agarose gel electrophoresis box, a common piece of laboratory equipment. Two RNA molecules (67 and 298 nucleotides long) transcribed in vitro from a truncated human beta-globin gene have been fingerprinted and analyzed by the modified technique. The resolution of the oligonucleotides in this procedure is comparable to that seen using the tank and is sufficient to separate relatively large oligonucleotides with similar base composition.

Base Sequence↗
PubMed1987

Dideoxy sequencing of double-stranded DNA from poly(A)-extended 3'-ends.

A simple method has been developed for sequencing double-stranded DNA by the chain termination method. The DNA to be sequenced is cut with a restriction enzyme that leaves a 3'-overhang which is extended by terminal deoxynucleotidyltransferase with limiting amounts of dATP. The sequencing reaction is then primed with an oligo(dT) primer which has a base pair "anchor" complementary to the overhang generated by the restriction enzyme. The method presented here eliminates the need for subcloning of the DNA or sequencing by chemical modification. Furthermore, sequences of more than 300 nucleotides are obtained from any 3'-overhanging restriction end.

Autoradiography↗
PubMed1990

Nucleic acid sequence analysis software for microcomputers.

It is clear that a computer-aided data control system is required for even small laboratories generating nucleic acid data. While the molecular biologist at many universities and large research institutions has access to mainframe computers and nucleic acid sequence analysis software, many find it more convenient to perform sequence analysis on microcomputers that are typically located within the investigator's laboratory and totally dedicated to sequence storage and analysis, in essence giving the investigator more personal control of analysis activities than is sometimes possible with shared mini- or mainframe computers. New programs are being written and released at an increasing rate to perform increasingly more complex and specialized analyses using small computer-based systems. This trend will undoubtedly continue, fueled by the need to manage the ever increasing quantity of sequence data.

Base Sequence↗
PubMed1990

A small-scale plasmid preparation yielding DNA suitable for double-stranded sequencing and in vitro transcription.

A small-scale plasmid preparation is described that is useful for a variety of procedures from double-stranded sequencing to in vitro transcription. No specialized equipment or reagents are required. The preparation of plasmid DNA does not require the use of RNase; instead the larger RNAs are precipitated with 2.5 M ammonium acetate. The resulting plasmid DNA is used routinely for double-stranded sequencing with the Klenow fragment of DNA polymerase and has been used for generating deletions with exonuclease III. In addition, the plasmid DNA has been used to generate transcripts with T7 RNA polymerase that translate well in reticulocyte lysate.

DNA↗
PubMed1990

A rapid, high resolution DNA sequencing gel system.

We have developed a simple method which significantly increases the efficiency of manual DNA sequencing. This method increases both the ease of gel preparation and the quality of fragment resolution. Our system involves (i) casting of gels horizontally, without sealing around the plates; (ii) the use of a self-forming buffer gradient to stack bands in the lower part of the gel; (iii) separation of the samples on two 0.2-mm-thick acrylamide gels (4.5 and 4%) with overlapping readings; (iv) "nonsmiling" electrophoresis with very simple, self-made electrophoresis stands; and (v) prior to exposure in situ dry fixation of the gel matrix to the glass support without previous covalent binding of the gel. On average, we are able to read from nucleotide position 50 to position 600 without ambiguity.

Acrylamides↗
PubMed1989

Glucocorticoid regulation of the rat cytochrome P450c (P450IA1) gene: receptor binding within intron I.

The actions of polycyclic aromatic hydrocarbons and glucocorticoids to regulate the expression of cytochrome P450c were investigated using cultured fetal rat hepatocytes. Cytochrome P450c mRNA content, determined by Northern blot analysis, was induced in cells treated with 1,2-benzanthracene from levels undetectable in untreated cells. When dexamethasone was included in the culture medium together with 1,2-benzanthracene there was a further 2-fold increase in the induction of cytochrome P450c mRNA. The concentration of dexamethasone required for a half-maximal increase in cytochrome P450c mRNA content was approximately 10(-9) M. By nuclear run-on transcription assays, treatment with 1,2-benzanthracene induced cytochrome P450c transcription 5.3-fold over untreated cells. In the presence of dexamethasone and 1,2-benzanthracene, there was a further 2-fold increase in cytochrome P450c transcription. Southwestern blotting and exonuclease footprinting methods have identified binding interactions of a purified glucocorticoid receptor fraction with portions of the cytochrome P450c gene within the first intron. Using a chimeric plasmid containing the first intron, the first exon, and 824 bp of 5'-flanking region of the cytochrome P450c gene, chloramphenicol acetyltransferase activity was induced in transfected HepG2 hepatoma cells by the addition of 1,2-benzanthracene. The addition of dexamethasone induced a further 2.2-fold increase in activity. Deletion of the first intron within the chimeric plasmid abolished responsiveness to dexamethasone. It is concluded that glucocorticoids act together with polycyclic aromatic hydrocarbons to increase the levels of cytochrome P450c expressed in the fetal rat hepatocyte, and that this action is mediated by the glucocorticoid receptor. A glucocorticoid responsive element, which binds the glucocorticoid receptor, has been identified within the first intron of the cytochrome P450c gene. These results suggest that glucocorticoids play a significant role in the response of the hepatic cytochrome P450c gene to xenobiotics.

Actins↗
PubMed1986

Expression of the fibrinogen genes in rat megakaryocytes.

A variety of evidence suggests that megakaryocytes synthesize fibrinogen and comparative immunochemical and structural studies indicate that fibrinogen produced in or associated with megakaryocytes may be different than fibrinogen produced in the liver. Two studies have reported that the gamma' chain, which is produced from the gamma chain gene by alternative splicing, is absent from fibrinogen produced in the megakaryocyte. Since there is only a single gene for each of the three fibrinogen chains the reported structural differences suggest different mechanisms for production of hepatic and megakaryocytic fibrinogen. We have begun an investigation of the varying mechanisms for expression of the fibrinogen genes by examining the structure of fibrinogen mRNA's in the two tissues. Fibrinogen mRNA's of identical length are found in both liver and megakaryocytes. Furthermore, despite the reported absence of the gamma' chain in platelet-associated fibrinogen, we have used a probe specific for the alternative spliced region of the gamma' mRNA to clearly demonstrate this chain in megakaryocyte mRNA. These studies indicate that the gamma' mRNA is either not translated in platelets or that the gamma' chain is unable to associated with the alpha and beta chains to form a mature molecule.

Animals↗