Structural analysis on the single-stranded genomic DNAs of the virus newly isolated from silkworm: the DNA molecules share a common terminal sequence

Summary Recently, a parvo-like virus was newly isolated from silkworm larvae and the two viral DNAs (VD1 and VD2) with different electro-mobilities were identified. We cloned the viral DNAs in a plasmid pUC119 and demonstrated that these two DNAs were not a bimorphic molecules though they shared a common terminal sequence of 53 nucleotides. In addition, the sequence at the 5 terminus of each strand of the viral DNA was located in inverted form at its 3 terminus. On the other hand, the nucleotide sequences of VD1 and VD2 were different from that of the Bombyx densovirus (Ina isolate) DNA.     

Rapidly evolving repetitive DNAs in a conservative genome: A test of factors that affect chromosomal evolution

The hypothesis that tandemly repeated DNA sequences may facilitate chromosomal rearrangements was tested by comparing a conservatively evolving karyotype of a bat species (Macrotus waterhousii) with data published for a rapidly evolving karyotype of an equid species (Equus zebra). Empirical data generated from the phylogenetic screening of rapidly evolving repetitive DNAs from approximately 0.1% of theM. waterhousii genome showed only one sequence that was repetitive inM. waterhousii but low in copy number or absent from the outgroupArtibeus jamaicensis. This compares to 34 such clones containing sequences which were repetitive inE. zebra but were low in copy number or absent from the outgroupCeratotherium simum. The bat sequence represents a single family of repeated sequences, whereas six families of sequences were identified inE. zebra. Southern blot analysis suggested that the sequence fromM. waterhousii is interspersed rather than tandemly repeated, as are the sequences inE. zebra. These data support the above hypothesis and suggest that species with conservatively evolving karyotypes have fewer numbers and families of rapidly evolving DNA sequences than do species such as the equids that possess a karyotype that is considered to have undergone rapid karyotypic evolution.     

Two novel mutations in the LDL receptor gene: common causes of familial hypercholesterolemia in a Spanish population

In our investigation of the LDL receptor gene in 30 Spanish patients, who were clinically diagnosed as heterozygous FH and were unrelated, we have applied single strand conformation polymorphism (SSCP) analysis and solid-phase sequencing. We identified two novel pathogenic mutations accounting for one third of the FH in this patient sample. Six patients were found to have a G to T substitution at nucleotide position 91 in exon 2 that results in a stop codon, E10X. Four patients were found to have a G deletion at nucleotide 518 in exon 4, causing a translational frameshift and a stop codon, 518delG. We have developed two polymerase chain reaction (PCR) based assays to detect easily these two mutations in all the available family members. We found fourteen E10X mutation carriers and eighteen 518delG mutation carriers. There was no statistically significant difference in mean lipid levels between carriers of these two mutations. Furthermore, haplotype analysis revealed that all E10X mutation carriers had the allele determined by TaqI-, Stul+, Avall+, Ncol- and all 518delG mutation carriers had the haplotype TaqI-, Stul+, Avall-, NcoI+. This indicates that both mutations may have been inherited from common ancestors, respectively.     

Developmental genetics of Coprinus cinereus: Genetic evidence that carpophores and sclerotia share a common pathway of initiation

Summary Five haploid monokaryons of the basidiomycete Coprinus cinereus were already known to be unable to form sclerotia (asexual resting structures) on the vegetative monokaryotic mycelium. Genetic analyses had shown that four distinct genes (symbolised scl were represented, all being recessive to their sclerotium-producing alleles. In the study reported, homoallelic dikaryons were constructed and the effect of the sclerotium-negative genes on carpophore formation investigated. In the homoallelic state these defective genes prevent the formation of both sclerotia and carpophores by the dikaryon. Thereby demonstrating that these two structures share a common pathway of initiation. It is also shown that the expression of effects of scl genes on carpophore maturation in heteroallelic dikaryons is subject to the influence of modifying genes. Possible modes of action of modifying genes and of the scl genes are discussed and a carpophore developmental pathway is presented.     

Molecular heterogeneity in mucopolysaccharidosis IVA in Australia and Northern Ireland: Nine novel mutations including T312S,a common allele that confers a mild phenotype

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive lysosomal storage disorder caused by a genetic defect in N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Previous studies of patients from a British–Irish population showed that the I113F mutation is the most common single mutation among MPS IVA patients and produces a severe clinical phenotype. We studied mutations in the GALNS gene from 23 additional MPS IVA patients (15 from Australia, 8 from Northern Ireland), with various clinical phenotypes (severe, 16 cases; intermediate, 4 cases; mild, 3 cases). We found two common mutations that together accounted for 32% of the 44 unrelated alleles in these patients. One is the T312S mutation, a novel mutation found exclusively in milder patients. The other is the previously described I113F that produces a severe phenotype. The I113F and T312S mutations accounted for 8 (18%) and 6 (14%) of 44 unrelated alleles, respectively. The relatively high residual GALNS activity seen when the T312S mutant cDNA is overexpressed in mutant cells provides an explanation for the mild phenotype in patients with this mutation. The distribution and relative frequencies of the I113F and T312S mutations in Australia corresponded to those observed in Northern Ireland and are unique to these two populations, suggesting that both mutations were probably introduced to Australia by Irish migrants during the 19th century. Haplotype analysis using 6 RFLPs provides additional data that the I113F mutation originated from a common ancestor. The other 9 novel mutations identified in these 23 patients were each limited to a single family. These data provide further evidence for extensive allelic heterogeneity in MPS IVA in British–Irish patients and provide evidence for their transmission to Australia by British–Irish migrants. Hum Mutat 11:202–208, 1998. © 1998 Wiley-Liss, Inc.     

Polymorphic sequences of the tyrosinase gene: allele analysis on 16 OCA1 patients in Japan indicate that three polymorphic sequences in the tyrosinase gene promoter could be powerful markers for indirect gene diagnosis

Since 1989, a large number of mutations of the tyrosinase gene, which result in oculocutaneous albinism (OCA), have been reported. However, approximately 15% of patients with tyrosinase-related OCA (OCA1) heterozygously carried an uncharacterized mutation, which presumably existed outside of the ordinarily examined area of the tyrosinase gene. In such cases, polymorphic sequence(s) of the tyrosinase gene might be useful to identify the OCA1 allele. In this study, we examined four polymorphic sequences of the tyrosinase gene in 16 patients with OCA1, their relatives, and 108 normally pigmented Japanese individuals. The results showed a complex dinucleotide repeat in the promoter region at −800 to −900 of seven different lengths, and a polythymidine sequence in the 3′ end of intron 2 of three different lengths. Polymerase chain reaction-restriction fragment length polymorphism analysis of two polymorphic sequences at −301 (C/T) and −199 (C/A) in the promoter region allows us to classify the tyrosinase gene into three groups. Using these polymorphic sequences, we could identify the OCA1 allele in more than 80% of cases in which the parents' genomic DNA was available. Three polymorphic sequences in the tyrosinase gene promoter are particularly useful for this purpose. Received: July 2, 2001 / Accepted: October 5, 2001     

Scanning method of identify the molecular heterogeneity of δ-globin gene especially in δ-thalassemias: Detection of three novel substitutions in the promoter region of the gene

A scanning strategy for the detection of δ-globin gene mutations and polymorphisms is presented. This procedure is based on the denaturing gradient gel electrophoresis (DGGE) of four different artificially amplified DNA fragments which cover the promoter, the exons, as well as IVS I of the reported gene. To estimate the efficiency and sensitivity of the proposed procedure, we analysed the appropriate controls of δ-thalassemic carriers, uncharacterised δ-thalassemias and cases with normal hematological phenotype, but slightly increased (up to 3.5%) HbA₂. DGGE results permitted the identification of δ-globin gene mutations and the polymorphism - 199 (T→C). Three novel base substitutions inside the promoter region of the gene [−65(A→G), −55(T→C), −36(C→A)], were also revealed. These changes are either linked in cis with other mutations or are responsible for thalassemias or for positive regulatory effect in δ-globin gene expression. The proposed experimental strategy consists of an accurate, rapid, safe and inexpensive screening procedure for establishing the molecular basis of δ-globin gene defects, suitable for the application for both research and diagnostics. Hum Mutat 9:465–472, 1997 © 1997 Wiley-Liss, Inc.     

Optional elements in the chloroplast DNAs of Chlamydomonas eugametos and C. moewusii: unidirectional gene conversion and co-conversion of adjacent markers in high-viability crossses

Unlike most polymorphic markers in the Chlamydomonas eugametos and Chlamydomonas moewusii chloroplast DNAs (cpDNAs), the C. moewusii 6- and 21-kb extra sequences and the C. eugametos-specific CeLSU ⋅ 5 intron are transmitted to all of the few viable progeny in reciprocal crosses between the two green algae. To determine whether this unidirectional transmission pattern is due to gene conversion or to selection for F1 hybrid survival, we followed the inheritance of the parental alleles at the loci featuring these three deletions/additions and at several other polymorphic cpDNA loci in zygospore clones derived from high-viability crosses. The great majority of the zygospore clones examined inherited exclusively the long alleles from the mt ^– parent at the loci containing the three optional cpDNA elements, but as expected, they preferentially inherited the markers from the mt ⁺ parent at most other loci. Our results therefore indicate that all three optional cpDNA sequences propagate themselves very efficiently by gene conversion in crosses between strains differing by the presence of these elements. The co-conversion tracts associated with these sequences are longer (>3 kb) than those previously reported for mobile elements spreading by gene conversion. Our results also revealed that less efficient gene conversion events occurred at two other cpDNA loci. Received: 12 February / 14 May 1996     

A ribosomal protein gene cluster is encoded in the mitochondrial DNA of Dictyostelium discoideum: UGA termination codons and similarity of gene order to Acanthamoeba castellanii

We sequenced a region of about 14.5 kb downstream from the ribosomal protein L11 gene (rpl11) in the mitochondrial DNA (54±2 kb) of the cellular slime mold Dictyostelium discoideum. Sequence analysis revealed that eleven ribosomal protein genes and six open reading frames (ORFs) formed a cluster arranged in the order: rpl11–orf189–rps12–rps7–rpl2–rps19–orf425–orf1740–rpl16–rpl14–orf188–rps14–rps8–rpl6–rps13–orf127–orf796. This order was very similar to that of homologous genes in Acanthamoeba castellanii mitochondrial DNA. The N-terminal region of ORF425 and the C-terminal region of ORF1740 had partial similarities to the S3 ribosomal protein of other organisms. The termination codons of rpl16 and orf188 were UGA, which has not hitherto been found in genes encoded in D. discoideum mitochondrial DNA. Received: 28 August 1997 / 2 January 1998     

Evolution of mitochondrial DNA in yeast: gene order and structural organization of the mitochondrial genome of Saccharomyces uvarum

We have determined the size, the restriction map and the gene order of the mitochondrial genome of the yeast Saccharomyces uvarum. Sequence analysis of the mitochondrial COXII gene confirmed the position of this yeast in the Saccharomyces cerevisiae-like group, near Saccharomyces cerevisiae and Saccharomyces douglasii. Most mitochondrial genes have been positioned on this approximately 57-kb long genome and three regions containing putative replication origins have been identified. The gene order of S. uvarum suggests that the mitochondrial genome of the S.cerevisiae-like yeasts could have evolved from an ancestral molecule, similar to that of S. uvarum, through specific genome rearrangements. Received: 22 April / 2 September 1997     

Mutational spectrum of the TSC1 gene in a cohort of 225 tuberous sclerosis complex patients: no evidence for genotype-phenotype correlation

Tuberous sclerosis complex is an inherited tumour suppressor syndrome, caused by a mutation in either the TSC1 or TSC2 gene. The disease is characterised by a broad phenotypic spectrum that can include seizures, mental retardation, renal dysfunction, and dermatological abnormalities. The TSC1 gene was recently identified and has 23 exons, spanning 45 kb of genomic DNA, and encoding an 8.6 kb mRNA. After screening all 21 coding exons in our collection of 225 unrelated patients, only 29 small mutations were detected, suggesting that TSC1 mutations are under-represented among TSC patients. Almost all TSC1 mutations were small changes leading to a truncated protein, except for a splice site mutation and two in frame deletions in exon 7 and exon 15. No clear difference was observed in the clinical phenotype of patients with an in frame deletion or a frameshift or nonsense mutation. We found the disease causing mutation in 13% of our unrelated set of TSC patients, with more than half of the mutations clustered in exons 15 and 17, and no obvious under-representation of mutations among sporadic cases. In conclusion, we find no support for a genotype-phenotype correlation for the group of TSC1 patients compared to the overall population of TSC patients.     

A mutation hotspot in the chloroplast genome of a conifer (Douglas-fir: Pseudotsuga) is caused by variability in the number of direct repeats derived from a partially duplicated tRNA gene

We determined the DNA sequence of a 2.7-kb cpDNA XbaI fragment from douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. RFLPs revealed by the 2.7-kb XbaI clone were observed to vary up to 1 kb among species within the genus Pseudotsuga and up to 200 bp among trees of P. menziesii. The polymerase chain reaction (PCR) allowed the locus of polymorphism to be identified, and the variable region was then sequenced in a second Douglas-fir tree, a single tree of a related species, Japanese Douglas-fir (P. japonica), and in a species lacking a mutation hotspot in the region, Pinus radiata (Monterey pine). The locus of polymorphism is characterized by hundreds of base pairs of imperfect, tandem direct repeats flanked by a partially duplicated and an intact trnY-GUA gene. The duplication is direct in orientation and consists of 43 bp of the 3 end of trnY and 25 bp of its 3 flanking sequence. Tandem repeats show high sequence similarity to a 27-bp region of the trnY gene that overlaps one end of the duplication. The two trees of Douglas-fir sequenced differed by a single tandem repeat unit, whereas these trees differed from the Japanese Douglas-fir sequenced by approximately 34 repeat units. Repetitive DNA in the Pseudotsuga cpDNA hotspot was most likely generated at the time of the partial trnY gene duplication and these sequences expanded by slipped-strand mispairing and unequal crossingover.     

Analysis of clinicopathologic features and gene mutations in gastrointestinal stromal tumor: a series of 58 patients

Background: In gastrointestinal stromal tumor (GIST), mutually exclusive gain-of-function mutations of c-kit and PDGFRα are associated with different mutation-dependent clinical features. We analyzed clinico-pathologic features and genotypes of GIST among patients in China. Methods: Adult patients with GIST in the stomach, small intestine, colorectum, or extra-gastrointestinal areas were enrolled in this study. These patients had been subjected to surgical resection without imatinib (Gleevec) treatment at the Cancer Hospital, Chinese Academy of Medical Sciences from January 2009 to January 2019. Samples were obtained for histopathologic examination. Mutations in c-kit and PDGFRα genes were analyzed by PCR and next generation sequencing (NGS). Clinico-pathologic characteristics of each gene were also analyzed. Results: A total of 58 GIST patients was enrolled in this study. In terms of genotypes, there were 51 (87.9%) c-kit mutations, 5 (8.6%) PDGFRα mutations, and 2 (3.4%) wild-type mutations. In terms of cell types, there were 40 cases (69.0%) with spindle cell type, 3 cases (5.2%) with epithelioid cell type and 3 cases (5.2%) with mixed spindle-epithelioid cell type. Among the 4 mutant forms of c-kit exon-11, the most common were point mutations in 16 cases (38.1%), deletion mutations in 13 cases (31.0%), insertion mutations in 4 cases (9.5%), and mixed mutations in 9 cases (21.4%). Based on risk grade classification of the National Institutes of Health (NIH), 3 cases (5.2%) were very-low risk, 9 cases (15.5%) were low risk, 19 cases (32.8%) were medium risk, and 23 cases (39.7%) were high risk. Significant differences in cell type were identified across different gene types (P = 0.022). Similarly, differences in tumor risk were found among different mutant forms of c-kit gene exon-11 (P = 0.039). Conclusion: With c-kit mutations, spindle cell type prevalence exceeded that of the epithelioid cell type and mixed spindle-epithelioid cell type. Spindle and mixed spindle-epithelioid cell types were the most prevalent in the category of PDGFRα mutations. In wild type cases, spindle and epithelioid cell types were the most common. A high risk of deletion and mixed mutations, and intermediate risk of point and insertion mutations were observed in c-kit exon-11 mutation type.     

Cloning of a yeast gene “in vivo” and its transfer as a part of an autonomously replicating unit: gene seduction

Summary Yeast diploids containing a chromosomally integrated episomal plasmid manifest the mitotic instability of the chromosomes with the plasmid. Probably as a results of the destabilization integrated plasmid may be excised out of the chromosome. It was found that the rescue of the plasmid may be irregular, with the taking up of adjacent chromosomal gene. Using an integrant with the plasmid integrated into chromosome I very near the ADE1 locus we cloned this gene in vivo by selecting rescued plasmid marker LEU2. The new plasmid has retained the LEU2 gene and the capacity to replicate autonomously. This plasmid was used to transfer the cloned ADEI gene from one cell to another by transformation and from one resident chromosome to another by integration.The phenomenon of irregular excision of an integrated episomal plasmid together with linked chromosomal gene(s) and transfer to other chromosomes or cells we propose to term Seduction.     

Clinical and molecular characterization of chronic hepatitis B in Albania: a country that is still highly endemic for HBV infection

Albania is a Mediterranean country, still with a high endemicity level of hepatitis B virus (HBV) infection. The chronic hepatitis B profile was characterized in this geographical area and used as a model to investigate the impact of endemicity level on the prevalence of the two major forms of chronic hepatitis B (HBeAg-positive and HBeAg-negative chronic hepatitis B). A cross-sectional study was conducted among 62 chronic hepatitis B patients consecutively admitted to the most important tertiary health care center for the diagnosis and treatment of liver disease in Albania. HBV-DNA was measured with an in-house PCR with a sensitivity of 10(4) copies/ml which uses primers encompassing the pre-core/core region. PCR products were subjected to sequencing and oligohybridization assay. Of the 62 patients, 75.8% had HBeAg-negative chronic hepatitis B. Genotype D was found in all 39 patients with detectable HBV viremia, for whom the heterogeneity of the region modulating HBeAg expression was assessed. Basic core promoter (BCP) mutations (1762/1764) were observed more often in anti-HBe-positive and older patients. In more than 90% of the HBeAg-negative chronic hepatitis B patients with detectable viremia, HBV that carries the G to A pre-core mutation at nucleotide 1896 was found. Patients with HBeAg-positive chronic hepatitis B were younger than HBeAg-negative chronic hepatitis B patients, and for symptomatic and asymptomatic liver-disease patients, the age of peak prevalence was at least 10 years lower for HBeAg-positive chronic hepatitis B patients. In conclusion, the virological and clinical pattern of chronic hepatitis B in Albania is similar to that observed in other Mediterranean countries; it seems to be independent of the HBV endemicity level.