Restriction Site Analysis Research Articles

Molecular evidence for multiple origins of hybridogenetic fish clones (Poeciliidae:Poeciliopsis).

Hybrid matings between the sexual species Poeciliopsis monacha and Poeciliopsis lucida produced a series of diploid all-female lineages of P. monacha-lucida that inhabit the Río Fuerte of northwestern Mexico. Restriction site analyses of mitochondrial DNA (mtDNA) clearly revealed that P. monacha was the maternal ancestor of these hybrids. The high level of mtDNA diversity in P. monacha was mirrored by similarly high levels in P. monacha-lucida; thus hybridizations giving rise to unisexual lineages have occurred many times. However, mtDNA variability among P. monacha-lucida lineages revealed a geographical component. Apparently the opportunity for the establishment of unisexual lineages varies among tributaries of the Río Fuerte. We hypothesize that a dynamic complex of sexual and clonal fishes appear to participate in a feedback process that maintains genetic diversity in both the sexual and asexual components.

Clinical and Molecular Heterogeneity in Hereditary β-Galactosidase Deficiency

Results of a molecular analysis of GM1-gangliosidosis and galactosialidosis in our laboratory are briefly reviewed. A common single base substitution was found in adult/chronic form of GM1-gangliosidosis among heterogeneous beta-galactosidase gene mutations, and restriction site analysis was successfully performed for diagnosis of homozygotes and heterozygotes. All adult galactosialidosis patients had a common mutation at a splice junction which caused skipping of an exon of the protective protein/carboxypeptidase gene. An artificial restriction site was introduced in this case and applied to diagnosis of this disease. The heterogeneous gene mutations were compared and correlated with phenotypic manifestations in these two diseases.

Rapid Screening of Mutant N-ras Alleles by Analysis of PCR-Induced Restriction Sites: Allele Specific Restriction Analysis (ASRA)

We have developed a rapid screening method for analysis of codon 12, 13 and 61 N-ras gene mutations, since these mutations have been observed in approximately 25% of patients with acute myeloid leukemia and myelodysplastic syndromes. The method, termed allele specific restriction analysis (ASRA), involves polymerase chain reaction amplification of DNA or RNA using a mismatched primer which introduces appropriately positioned base substitutions in N-ras and creates a restriction site provided the adjacent sequence is normal. Simultaneous analysis of codons 12 and 61 is also possible by the use of a multiprimer reaction mixture. Resistance of the amplified product to digestion indicates the presence of a mutation in the original template. Since ASRA allows simultaneous analysis of mutant and wild type sequences in DNA and RNA, an estimate of the ratio of gene copies and relative expression of N-ras alleles can be obtained for heterozygous individuals.

The Use of Chloroplast DNA to Assess Biogeography and Evolution of Morphology, Breeding Systems, and Flavonoids in Fuchsia sect, Skinnera (Onagraceae)

Fuchsia sect. Skinnera, the only Old World group in the genus, comprises three species in New Zealand and one in Tahiti. These species include a tree, a large shrub, a climbing-scandent shrub, and a creeping, barely woody plant. The section is extremely distinct from the New World species of Fuchsia, and several sharply contrasting models depicting the evolutionary history of these species and the character state changes associated with habit, breeding systems, and chemistry have been proposed. A chloroplast DNA restriction site analysis of sect Skinnera and two outgroup sections was performed, generating a single tree depicting maternal relationships. This tree is congruent with one previously proposed model and demonstrates that the rare F. procumbens of New Zealand is the sister group to all other species in the section, while F. cyrtandroides of Tahiti is the sister taxon of the two remaining New Zealand species. Based on the chloroplast DNA analysis, a critical reevaluation was made of character state changes in the section relating to morphology, breeding systems, and flavonoid compounds. Phylogenetic analysis of plants using chloroplast DNA (cpDNA) is increasingly providing detailed and often unexpected evidence of evolutionary relationships at many taxonomic levels (Jansen and Palmer 1987a, 1988; Palmer 1986; Palmer and Zamir 1982; Palmer et al. 1983, 1985, 1988; Rieseberg et al. 1988; Smith and Sytsma 1990; Soltis et al. 1988; Sytsma 1990; Sytsma and Gottlieb 1986a, 1986b; Sytsma and Schaal 1985; Sytsma and Smith 1988; Sytsma et al. 1990). A major strength of cpDNA restriction fragment analysis is that it provides numerous, independent molecular characters that can often rigorously define monophyletic lineages. The utility of cpDNA, as well as other molecular markers, in phylogenetic studies can involve two separate steps. The first consists of defining monophyletic lineages, refining estimates of historical genealogies, and changing taxonomic groupings as needed; that is, addressing questions about phylogenetic relationships. The second step involves the application of the resulting cladogram or phylogeny to questions such as the evolution of morphology, breeding systems, and biogeography; that is, addressing questions about character evolution (where, when, and how the character states arose). Through this secondary application, molecular phylogenetics is having a considerable impact on many aspects of systematics, evolution, genetics, and ecology. This two-step process is especially crucial and informative when dealing with groups of species that show tremendous divergence and radiation in morphology, chemicals, and ecology (Sytsma 1990). Analysis of non-molecular characters in such groups can often be phylogenetically uninformative or even misleading because of the operation of strong selection and resulting homoplasy, difficulty in ordering or even polarizing character states, the high number of autapomorphies, and the lack of well defined synapomorphies. An exemplary model of a lineage that has undergone extraordinary radiation but is difficult to interpret in an evolutionary sense is Fuchsia sect. Skinnera. This section has been the focus of several competing hypotheses concerning the evolution and relationships of component taxa and the nature of changes in habit, morphology, floral biology, flavonoids, and distribution. The genus Fuchsia, distinctive in the Onagraceae by its fleshy fruits, corolloid calyx, and unspecialized features in floral and vegetative

Identification and cloning of a hemin storage locus involved in the pigmentation phenotype of Yersinia pestis

The temperature-dependent absorption of sufficient exogenous hemin or Congo red to form pigmented colonies of Yersinia pestis has been termed the pigmentation phenotype (Pgm+). Spontaneous mutation to a Pgm- phenotype results in the loss of a number of divergent physiological characteristics, including the ability to store hemin and to bind Congo red at 26 degrees C. In this study, we generated and isolated transposon insertion mutants that are hemin storage negative (Hms-) and therefore unable to form pigmented colonies. These mutations are due to single mini-kan insertions within a 19.5-kilobase (kb) SalI fragment of chromosomal DNA. Restriction site analysis of eight mutants identified a minimum of six potentially different insertion sites spanning an approximately 10-kb hemin storage (hms) locus. The 19.5-kb SalI fragment (containing approximately 18 kb of Y. pestis DNA and the mini-kan insert) was cloned from one of these mutants, KIM6-2012. By using this cloned fragment as a DNA probe, the mechanism of spontaneous mutation to a Pgm- phenotype was identified as a massive deletion event. The deletion spans at least 18 kb of genomic DNA in spontaneous Pgm- mutants from nine separate strains of Y. pestis. DNA adjacent to the mini-kan insert was used to identify a clone containing a wild-type hms locus. A spontaneous Pgm- mutant of Y pestis KIM containing this clone exhibits an Hms+ phenotype. The hms::mini-kan mutations and cloned wild-type hms locus generated in this study will greatly aid in identifying the function of hemin storage in Y. pestis.

Molecular basis of HbE-β-thalassemia and the origin of HbE in northeast Thailand: Identification of one novel mutation using amplified DNA from buffy coat specimens

Amplification of DNA via polymerase chain reaction directly from a small amount of a buffy coat fraction was used to study the molecular basis of HbE-β-thalassemia in the northeastern Thai population. Eight different mutations including the new one causing a β o-thalassemia phenotype were detected. This novel mutation is an amber mutation at codon 26, which occurs at the same position as that of HbE: the most common hemoglobin variant in Southeast Asian countries. A pitfall in detection of the HbE mutation by restriction enzyme analysis was pointed out and differential diagnosis of the HbE mutation and the novel one by using allele specific oligonucleotide probes were described. Analysis of polymorphic restriction sites in the β-globin gene cluster containing the β E gene revealed two previously undescribed haplotypes in the Southeast Asian populations, which provide evidence for the multiple origins of β E gene in Southeast Asian populations.

Subfamilial Phylogenetic Relationships of the Bromeliaceae: Evidence from Chloroplast DNA Restriction Site Variation

Restriction-site analyses of the chloroplast genomes of members of the Bromeliaceae were conducted to explore subfamilial phylogenetic relationships. Eighteen restriction-site muta- tions and one large (1.8-kb) length mutation were found to be variable at the family and subfamily levels. Cladistic analyses of these data supported the following hypotheses: 1) Bromelioideae and Pitcairnioideae are sister taxa and the latter may be paraphyletic; 2) Tillandsioideae s. str. and Bromelioideae are each monophyletic; 3) Tillandsioideae s. str. is the sister taxon of the remainder of the family; and 4) Glomeropitcairnia is phylogenetically distinct from the remainder of Tilland- sioideae. These analyses suggest that the primarily terrestrial Pitcairnioideae are not basal in the family even though the terrestrial habit apparently is ancestral in Bromeliaceae. Chloroplast DNA data also support the contention that some morphological characters have evolved independently several times in this large, ecologically complex family.

Aspartic acid at position 57 of the HLA-DQβ chain is not protective against insulin-dependent diabetes mellitus in Japanese people

Insulin-dependent diabetes mellitus (IDDM) in Caucasians is closely associated with the HLA-DQ gene, especially the residue 57 of the DQβ chain. Aspartic acid at this position provides protection against IDDM, and substitution of this residue by alanine, valine or serine increases susceptibility to IDDM. To determine whether this is a common feature of IDDM in different ethnic groups, we studied DQB1 DNA in Japanese patients with IDDM by polymerase chain reaction and non-radioactive restriction site analysis. In contrast to Caucasian patients with IDDM, most Japanese patients with IDDM possessed at least one aspartic acid at position 57 of DQβ. This finding strongly suggests that aspartic acid at position 57 of DQβ does not protect the Japanese from IDDM.

Two steroid 15α-hydroxylase genes and a homologous gene family in mice

Steroid 15α-hydroxylase (P450 15α) activity is concomitant with the expression of two types of mRNA in the mouse liver. Two discrete genes, designated 15 α oh-1 and 15 α oh-2, that encode the two mRNAs were recovered from total genomic libraries of the inbred mouse strains 129/J and C57B1/6J and identified by cDNA hybridization, restriction-site analysis and partial nucleotide sequence. Both genes are approx. 9 kb long and share significant homology, including flanking regions, over a region of at least 30 kb. The two distinct 15 α oh genes are members of a larger family of homologous genes and/or pseudogenes of unknown function. The most extensive sequence homology among family members is in the 3′ portion of the gene with progressively less homology toward the 5′ end. The far 5′ portions of 15 α oh-1 and 15 α oh-2 are very similar to one another but there is no observed homology with other genes of the family. The two 15 α oh genes and the homologous family have been localized to mouse chromosome 7 by somatic cell hybrid mapping. Analysis of a restriction fragment length polymorphism in recombinant inbred mice shows a close linkage of 15 α oh-1 and 15 α oh-2 with the Coh locus.

Molecular cloning of two west African HIV2 isolates that replicate well in macrophages

HIV2 strains were isolated from a Gambian with neuro-AIDS (HIV2 D194) and from an asymptomatic Ghanian (HIV2 D205). Like HIV1 biological subtype c, both isolates grew slowly and induced few or no syncytia, but eventually produced high levels of particle-associated reverse transcriptase (RT) in cultures of fresh peripheral blood lymphocytes. Each produced even higher levels of RT in fresh human macrophages, especially HIV2 D194, where maximal RT values of 1,800,000 cpm/ml supernatant of approximately 30,000 cells were measured. The viruses were molecularly cloned after a single passage in culture. Restriction-site analysis showed heterogeneity within each isolate. Nucleotide sequence analysis of HIV2 D194 revealed that, genetically, it is a member of the prototypic HIV2 family, displaying 12% divergence vs. HIV2 ROD and HIV2 NIHZ. In contrast, HIV2 D205 is the most highly divergent HIV2 strain yet described: it is equidistant in relation between the known HIV2 strains and the SIV MAC isolates (23–24 % nucleotide sequence divergence).

Chloroplast-DNA and Allozymic Variation in Diploid and Autotetraploid Heuchera grossulariifolia (Saxifragaceae)

Diploid and autotetraploid populations of Heuchera grossulariifolia occur throughout mountainous regions of the Pacific Northwest. Controlled greenhouse crosses indicated that the two cytotypes are largely reproductively isolated. Fourteen diploid and 11 tetraploid populations were analyzed electrophoretically. Individual tetraploid plants expressed up to four alleles per isozyme locus, and tetraploid populations had significantly higher levels of heterozygosity than diploids. Mean observed heterozygosity was 0.159 for tetraploid populations and 0.058 for diploid populations. The patterns of allelic distribution between cytotypes suggested multiple origins of autotetraploids. This hypothesis was supported by restriction-site analysis of chloroplast-DNA (cpDNA) variation which indicated that there had been at least three independent origins of tetraploids. Electrophoretic data, in conjunction with a cpDNA-based phylogeny and geographic distribution of populations, suggest that autopolyploid populations evolved several times as migration of diploids occurred down river systems. This study further supports the contention that autopolyploidy can be a common and successful speciation process in some groups of plants.

CHLOROPLAST-DNA AND ALLOZYMIC VARIATION IN DIPLOID AND AUTOTETRAPLOID HEUCHERA GROSSULARIIFOLIA (SAXIFRAGACEAE).

Diploid and autotetraploid populations of Heuchera grossulariifolia occur throughout mountainous regions of the Pacific Northwest. Controlled greenhouse crosses indicated that the two cytotypes are largely reproductively isolated. Fourteen diploid and 11 tetraploid populations were analyzed electrophoretically. Individual tetraploid plants expressed up to four alleles per isozyme locus, and tetraploid populations had significantly higher levels of heterozygosity than diploids. Mean observed heterozygosity was 0.159 for tetraploid populations and 0.058 for diploid populations. The patterns of allelic distribution between cytotypes suggested multiple origins of autotetraploids. This hypothesis was supported by restriction-site analysis of chloroplast-DNA (cpDNA) variation which indicated that there had been at least three independent origins of tetraploids. Electrophoretic data, in conjunction with a cpDNA-based phylogeny and geographic distribution of populations, suggest that autopolyploid populations evolved several times as migration of diploids occurred down river systems. This study further supports the contention that autopolyploidy can be a common and successful speciation process in some groups of plants.

Molecular approaches to the study of plant biosystematics

Genetic relationships among organisms can be estimated from the pattern of DNA sequence change between hereditary molecules. The methods of molecular biology are increasingly being employed in systematic and evolutionary research to study genetic relationships and phylogeny. The investigator is faced with a variety of choices in initiating research in 'molecular biosystematics'. First, a gene or genome that provides a level of genetic resolution appropriate for the materials under study must be selected. Common choices in plants include the chloroplast genome (cpDNA) or components of the chloroplast genome, the nuclear ribosomal RNA genes (rDNA), or nuclear-encoded, single-copy genes. A second consideration is that several methods can be employed to provide direct or indirect measures of DNA sequence divergence. One widely used method determines genetic divergence based on restriction site changes. Restriction site analyses typically require some knowledge of the physical map of the DNA molecule under study. Empirical studies indicate that restriction site analyses of cpDNA provide good resolution for systematic investigations at or below the family level. A second method that is increasing in importance is DNA sequencing. Until quite recently, DNA sequencing required the molecular cloning of the gene or DNA fragment of interest and the implementation of a 'sequencing strategy' for the production of overlapping sequence runs. Two related technological developments have overcome both of these requirements, when the goal is the repetitive sequencing of a specific gene from a number of taxa. It is now possible to synthesise synthetic oligonucleotides that can be used to prime dideoxy sequencing at virtually any point in the gene of interest, based on previously determined sequence information. The second major innovation is the invention of the polymerase chain reaction technique (PCR). The PCR method permits the direct amplification of DNA fragments from heterogeneous DNA mixtures and thereby circumvents the molecular cloning requirement. Application of the PCR method to chloroplast genes, together with direct primer-mediated sequencing, provides a method for obtaining large data sets. CpDNA sequence data are especially valuable at or above the family level and provide a powerful means of resolving genetic relationships at the deepest levels of plant evolution.

A novel C-T transition within the distal CCAAT motif of the Gγ-globin gene in the Japanese HPFH: implication of factor binding in elevated fetal globin expression

Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by the continued expression of the fetal globin gene in adulthood. Both deletional and nondeletional forms have been described. We studied one Japanese family with two different nondeletional forms of HPFH. Analysis of polymorphic restriction sites in the beta-globin gene cluster suggested that one affecting both G gamma and A gamma globin expression in two members of the family could be associated with unknown conditions not linked to the beta-globin gene loci. Characterization by the polymerase chain reaction (PCR) of another form producing a G gamma-HPFH phenotype in two other members demonstrated a novel C-T transition at the nucleotide -114 within the distal CCAAT motif of the G gamma-globin gene. Using gel retardation assays on various nuclear extracts, we also demonstrated that this novel mutation abolishes the binding of the ubiquitous CCAAT binding factor, CP1 to the distal CCAAT motif of the gamma-globin gene but does not affect the binding of any erythroid specific factor, thereby suggesting a possible role for CP1 in the developmental regulation of fetal globin expression.

Genetic Evidence for Allopolyploidy in the Neotropical Fern Hemionitis pinnatifida (Adiantaceae) and the Reconstruction of an Ancestral Genome

Genetic evidence from enzyme electrophoresis and restriction site analyses of cpDNA was employed to explore the origin of the tetraploid Hemionitis pinnatifida by determining the role of diploid species of Hemionitis in contributing to the tetraploid genome. The results clearly supported the hypothesis that H. pinnatifida is an allotetraploid with H. palmata as one of its diploid progenitors. The other diploid parent was not identified among the existing species of Hemionitis and is either extinct or exceedingly rare. Genetic identity analyses compared the extant diploid genomes with the divergent diploid genomes combined in tetraploids. One of these divergent genomes was shown to be a good approximation of that of H. palmata, and the other is a reconstruction of that of the missing diploid parent. These analyses demonstrated that the missing ancestor was genetically more similar to H. palmata than either of these species is to the other diploid species surveyed. Such greater genetic similarity may have been important in allowing hybridization between the two diploid progenitors of H. pinnatifida. The data also suggest that tetraploid individuals may have been synthesized de novo at least five times among the three populations surveyed.

Molecular cloning of two west African human immunodeficiency virus type 2 isolates that replicate well in macrophages: a Gambian isolate, from a patient with neurologic acquired immunodeficiency syndrome, and a highly divergent Ghanian isolate.

Human immunodeficiency virus type 2 (HIV-2)-related viruses were isolated from a Gambian dying of exclusively neurological disease (HIV-2D194) and from an asymptomatic Ghanian (HIV-2D205). Both strains exhibited properties of HIV-1 biological subtype c: they grew slowly and induced few or no syncytia but eventually produced high levels of particle-associated reverse transcriptase in cultures of fresh peripheral blood lymphocytes, and they established stable infection of T-lymphoma (HUT-78) and monocytic (U937) cell lines. Each produced even higher levels of reverse transcriptase when fresh human monocytes/macrophages were used as target cells. The viruses were molecularly cloned after a single passage in culture, in order to minimize in vitro selection of subtypes present in vivo. Restriction-site analysis showed heterogeneity within each isolate. Nucleotide sequence analysis of a portion of the HIV-2D194 genome revealed that it is a member of the prototypic HIV-2 family, displaying 13% divergence versus HIV-2ROD and HIV-2NIHZ, as compared to 9% divergence between HIV-2ROD and HIV-2NIHZ. In contrast, HIV-2D205 is the most highly divergent HIV-2 strain yet described: it is equidistant in relation between the known HIV-2 strains and the simian immunodeficiency virus isolates from rhesus macaque monkeys (23-25% divergence).

Plasmid DNA from the acetotrophic methanogen Methanosarcina acetivorans.

Nine acetotrophic and three methylotrophic strains of methane-producing bacteria were screened for the presence of plasmid DNA. Plasmids were detected in three marine isolates, including Methanosarcina acetivorans. All three plasmids appeared to be similar based on size and restriction site analyses. The plasmid from M. acetivorans, designated pC2A, was approximately 5.1 kilobase pairs in size and was estimated to be present in a low copy number of six plasmids per genome. Multimers were also observed. A restriction map was constructed. The function of this plasmid is cryptic.

Retropseudogenes for human chromosomal protein HMG-17

The human genome contains multiple copies of sequences homologous to the cDNA coding for non-histone chromosomal protein HMG-17. To study the mechanism of generation and dispersion of the HMG-17 multigene family a human genomic library was screened and 70 clones isolated and studied by Southern transfer and restriction site analysis. The results suggest that most of the clones contain unique sequences. Sequence analysis of two genomic clones indicates that they contain elements typical of processed retropseudogenes. Even though both sequences contained open reading frames the sequences lacked introns, were flanked by short, direct repeats and lacked elements associated with functional genes. The sequences of the two pseudogenes were 85 % homologous to each other and each was 90% homologous to the human cDNA. Based on the sequence difference in the open reading frame between the pseudogenes and the cDNA it can be estimated that the sequences arose approximately ten million years ago from a common precursor. The present paper, which is the first study on genes coding for this nucleosomal binding protein, indicates that the HMG-17 multigene family is the largest known human retropseudogene family.

Cloning of Streptomyces griseus and Streptomyces lividans genes for glycerol dissimilation

Streptomyces lividans gyl DNA (for glycerol utilisation) was cloned by complementation of a Streptomyces coelicolorgyl mutant. Restriction mapping showed that the cloned DNA was highly homologous (perhaps 99%) to S. coelicolor gyl DNA. Using phage-mediated mutational cloning, an internal fragment of the S. coelicolor gyl operon was used to generate a gyl mutant of S. lividans, which subsequently served as recipient in the cloning of gyl DNA from S. griseus. A 7.5-kb SstI-generated fragment of S. griseus DNA was obtained which, as judged by analysis of restriction sites, was only perhaps 87 % homologous with the S. coelicolor gyl operon. The cloned S. griseus DNA appears to contain intact gylA and gylB genes and probably also an upstream gene related to the putative gyl regulatory ‘0.9-kb’ gene of S. coelicolor. Cloning of the fragment on a high-copy-number vector in S. lividans did not lead to high levels of the enzymes encoded by gylA and gylB. The S. griseus gylA and gylB genes were not detectably expressed in Escherichia coli glp mutants.

Tolerated variations in a genome: the case of closely related Bacillus phages PZA, φ29 and φ15 — a review

Restriction-site analysis was used to estimate the relationship of bacteriophages PZA, φ29 and φ15. Complete nucleotide sequences of PZA and φ29 genomes were compared and tolerated variations were assessed. Most of the base-pair changes are silent nucleotide substitutions in the third position of codons but amino acid changing substitutions are also observed. The terminal portions of the phage genomes diverged faster than their central parts. Gene mutations in phage PZA were induced by hydroxylamine and their frequency was compared with the evolutionary mutability.