Restriction Endonuclease Cleavage Analysis Research Articles

A Partial Phenotype of adFNDI Related to the Signal Peptide c.55G>A Variant of the AVP Gene

The autosomal dominant familial form of neurohypophyseal diabetes insipidus (adFNDI) is a rare inherited endocrine disorder characterized by hypotonic polyuria, severe thirst and polydipsia, which results from a deficient neurosecretion of the antidiuretic hormone, also known as arginine vasopressin (AVP). To date, adFNDI has been linked to more than 70 different heterozygous point mutations of the 2.5 kb AVP gene, encoding the composite precursor protein of AVP. A minority of disease-causing mutations, such as the common c.55G>A variant, are predicted to affect amino acid residues close to the signal peptide (SP) cleavage site, and result in abnormal post-translational processing and intracellular trafficking of AVP precursors exerting neurotoxic activity on vasopressinergic magnocellular neurons. Generally, SP variants cause a gradual decline in the neurohypophyseal secretion of AVP in small children, although a wide variability in clinical onset and severity of manifestations has been reported. For the first time, we describe a kindred from Calabria (Southern Italy) with adFNDI and document a partial clinical phenotype in one female young adult member of the family. Methods: A young adult woman was subjected to clinical, neuroradiological and genetic assessments for a mild, adolescent-onset, polyuric state at our Endocrinology Unit. Her family medical history revealed an early-onset (<12 years of age) occurrence of polyuria and polydipsia, which was successfully managed with high doses of oral desmopressin, and a typical adFNDI inheritance pattern that was seen over three generations. Results: In the index patient, the extensive hypertonic dehydration during fluid deprivation test elicited a prompt elevation of urine osmolality and diuresis contraction, indicative of a partial adFNDI phenotype. Diagnosis was confirmed by concordant hormonal tests and magnetic resonance imaging (MRI) evidence of a reduced hyperintense signal of the neurohypophysis, which was regarded as compatible with the depletion of the vasopressinergic magnocellular neurons. Direct DNA sequencing and restriction enzyme cleavage analysis revealed that a heterozygous c.55G>A transition, predicting a p.Ala19Thr replacement in the C-terminal region of SP, was the cause of adFNDI in the investigated kindred. Conclusions: The identification of the genetic cause of aFNDI in this Calabrian kindred provides further information and confirms the wide variability of disease onset and severity of manifestations related to SP variants of the AVP gene, supporting the need for genetic testing in all patients with familial occurrence of polyuria, regardless of their clinical and radiological phenotype. Even though sexual differences in the antidiuretic responses are documented, it is unclear whether female gender would attenuate clinical disease progression in the presence of a pathogenic c.55G>A mutation.

The human insulin gene and diabetes mellitus.

The recombinant DNA technology is a useful tool to characterize the insulin gene and adjacent areas. A highly polymorphic region near the human insulin gene was detected and its possible relation to certain types of diabetes mellitus is discussed. In three cases the synthesis of a structurally abnormal insulin of lower biological activity leads to hyperglycemia. These mutant insulin gene sequences can be identified with the help of restriction endonuclease cleavage analysis. Insulin gene expression is regulated mainly at translational level. Elements near or within the insulin gene seem to be required for a sufficient and cell-specific expression. But the role of the polymorphic locus is not yet clear. First results on gene transplantation by administration of liposome entrapped insulin gene sequences are surprising, but at this time only of speculative value for medical research.

Spatial genetic subdivision between northern Australian and southeast Asian populations of Pristipomoides multidens: a tropical marine reef fish species

Nucleotide sequence polymorphism in the left domain of the control region of the mitochondrial genome of over 700 goldband snapper ( Pristipomoides multidens) was surveyed using both direct sequencing and innovative restriction enzyme cleavage analysis techniques. Southeast Asian populations were sampled adjacent to western Irian Jaya, northern Papua New Guinea and western Timor. Six Australian populations were sampled from adjacent to Exmouth in western Australia to Weipa in the northern Gulf of Carpentaria. The results show that significant genetic structure occurs among Indonesian and Australian waters along national boundaries; 14% of the total molecular variance among restriction site haplotypes was due to genetic distinction between Indonesian and Australian samples. Several lines of evidence suggest that gene flow does not occur freely along the northern and western Australian coastline, particularly on the northwestern Kimberley coast. Australian fisheries managers need to be alerted to the possibility of at least one genetically distinct population of this important commercial species which should be protected from over-harvesting that may otherwise lead to localised extinction and the erosion of genetic diversity. There is no convincing biological argument for the observed genetic disjunction in the Kimberley area. It may be due to the combined effect of past sea-level changes, sampling error or patterns of exploitation.

Evaluation of a new general primer pair for rapid detection and differentiation of HSV-1, HSV-2, and VZV by polymerase chain reaction.

The polymerase chain reaction (PCR) enables rapid and sensitive detection of VZV and HSV DNA and its efficiency depends mainly on the choice of the primers. Primers should hybridize to conserved DNA sequences within the viral genomes in order to avoid unreliable amplification due to DNA sequence variation between different strains. The aim of the study was to design and to evaluate a general primer pair which permits fast and reliable detection of HSV and VZV. The genes UL 15 of HSV and UL 42 of VZV share the highest degree of homology within the two genomes. We designed a primer pair (GPHV-RU) which hybridizes to these genes. The genetic variability of amplified sequences from clinical specimens was analyzed by restriction enzyme cleavage analysis and by temperature gradient SSCP analysis (TG-SSCP). PCR with GPHV-RU amplified viral sequences from all analyzed specimens (25 x VZV, 10 x HSV-1, 5 x HSV-2) obtained from patients with clinical evidence of HSV or VZV infection. Restriction enzyme cleavage analysis with Hpa II further permitted reliable distinction between VZV, HSV-1, and HSV-2. Analysis of the heterogeneity of the amplified sequences by restriction enzyme cleavage and by TG-SSCP demonstrated no variability between the analyzed clinical specimens of VZ and of HSV-2 and only one differing TG-SSCP-pattern within the HSV-1 isolates. The results suggest that detection of HSV and VZV using the new primer pair GPHV-RU should give reliable results as the amplified sequences show little genetic variability within clinical isolates of HSV-1/2 and VZV.

Evaluation of a new general primer pair for rapid detection and differentiation of HSV‐1, HSV‐2, and VZV by polymerase chain reaction

The polymerase chain reaction (PCR) enables rapid and sensitive detection of VZV and HSV DNA and its efficiency depends mainly on the choice of the primers. Primers should hybridize to conserved DNA sequences within the viral genomes in order to avoid unreliable amplification due to DNA sequence variation between different strains. The aim of the study was to design and to evaluate a general primer pair which permits fast and reliable detection of HSV and VZV. The genes UL 15 of HSV and UL 42 of VZV share the highest degree of homology within the two genomes. We designed a primer pair (GPHV-RU) which hybridizes to these genes. The genetic variability of amplified sequences from clinical specimens was analyzed by restriction enzyme cleavage analysis and by temperature gradient SSCP analysis (TG-SSCP). PCR with GPHV-RU amplified viral sequences from all analyzed specimens (25×VZV, 10×HSV-1, 5×HSV-2) obtained from patients with clinical evidence of HSV or VZV infection. Restriction enzyme cleavage analysis with Hpa II further permitted reliable distinction between VZV, HSV-1, and HSV-2. Analysis of the heterogeneity of the amplified sequences by restriction enzyme cleavage and by TG-SSCP demonstrated no variability between the analyzed clinical specimens of VZV and of HSV-2 and only one differing TG-SSCP-pattern within the HSV-1 isolates. The results suggest that detection of HSV and VZV using the new primer pair GPHV-RU should give reliable results as the amplified sequences show little genetic variability within clinical isolates of HSV-1/2 and VZV. © 1996 Wiley-Liss, Inc.

Molecular cloning and RARE cleavage mapping of human 2p, 6q, 8q, 12q, and 18q telomeres.

Large terminal fragments of human chromosomes 2p, 6p, 8q, 12q, and 18q were cloned using yeast artificial chromosomes (YACs). RecA-assisted restriction endonuclease (RARE) cleavage analysis of genomic DNA samples from II unrelated individuals using YAC-derived probes confirmed the telomeric localizations of the half-YACs studied. The cloned fragments provide telomeric closure of maps for the respective chromosome arms and will supply the reagents needed for analyzing and sequencing these distal subtelomeric regions.

Demonstration of either endogenous recurrence or exogenous reinfection by restriction endonuclease cleavage analysis of herpes simplex virus from patients with recrudescent genital herpes.

By using restriction endonuclease (RE) cleavage analysis, either endogenous recurrence or exogenous reinfection of herpes simplex virus (HSV) was clarified in 45 male and 20 female subjects with recrudescent genital herpes. All of the plural (two to ten) isolates from 63 (205 isolates) out of 65 subjects (97%) were HSV-2. Two isolates from only one of 65 subjects (1.5%) were HSV-1, and they showed the same RE profile. In addition, an HSV-1 and five HSV-2 isolates were obtained from the remaining one female patient (1.5%), indicating that an exogenous HSV-1 strain has been reinfected during HSV-2 recrudescences. HSV-2 isolates were furthermore classified into genotypes of HSV-2 using 16 different RE markers with five REs. Two hundred and ten HSV-2 isolates from 64 subjects were classified into 27 distinct genotypes, in which some predominant genotypes and seven new genotypes were found. Plural HSV-2 isolates obtained from 63 out of 64 subjects, including one subject from whom an HSV-1 and five HSV-2 strains were isolated, were classified into the same genotypes, indicating that they may be regarded as recrudescent genital herpes by the reactivation of the same endogenous strain. However, the RE profiles of two HSV-2 strains from the remaining one subject were different. Thus, it was finally found that only two out of 65 subjects (3%) were reinfected with exogenous strains. These results lead to the conclusion that almost all recrudescent genital herpes are due to the reactivation of an initially infected HSV-2 strain, and are occasionally due to reinfection with distinct HSV strains of either the same or a different type.

Molecular analysis of a novel subtelomeric repeat with polymorphic chromosomal distribution

DNA from a 50-kb yeast artificial chromosome (YAC) containing one human telomere was characterized. Cloned sequences from the centromeric end of this YAC (designated yRM2001) localized to several human chromosomes by somatic hybrid panel mapping. The telomeric end of the YAC contained both (TTAGGG)n sequences and the previously characterized TelBam3.4 subterminal repeat element. A novel low-copy repeat element (designated HC1103) mapped 19 kb from the telomeric end of the YAC. This repeat was shown by fluorescence in situ hybridization to be present in several subtelomeric regions (3q, 12p, 15q, 19p, and 20p) and at an interstitial site (2q13-->q14) in all individuals studied, but to be polymorphically distributed at several other telomeres. The YAC vector-insert EcoRI cloning site was positioned 50 kb to 70 kb from chromosome termini in human genomic DNA using RecA-assisted restriction endonuclease (RARE) cleavage analysis. Our results suggest that the DNA segment cloned in yRM2001 contains a novel block of low-copy DNA consistently present at some human telomeres, but polymorphically distributed at others.

Re‐evaluation of a case of progressive multifocal leukoencephalopathy previously diagnosed as simian virus 40 (SV40) etiology

A case of progressive multifocal leukoencephalopathy (PML) reported previously to be of simian virus (SV40) etiology was re-evaluated. The supernatant from a 10% homogenate of brain material was inoculated into African green monkey kidney cells and BSC-1 cells which are permissive for SV40. However no cytopathic effect (CPE) developed and no virus was isolated. The brain supernatant agglutinated human group O erythrocytes and contained 5120 units/mL. The Hirt supernatant from the brain contained three DNA bands corresponding to forms I, II and III of circular double-stranded viral DNA. Restriction endonuclease cleavage analysis revealed that this viral DNA was different from SV40 DNA, but similar to JC virus DNA. After cloning of this viral DNA into pBR322 at the BamHI site, DNA homology of this virus and of SV40 was investigated. Cloned DNA from the brain hybridized with all the HpaI/EcoRI fragments of the SV40 genome at the effective temperature of Tm -50 degrees C [corrected]. At Tm -28 degrees C, however, the cloned DNA hybridized with only HpaI/EcoRI fragment B of the SV40 genome. In contrast to this, JC virus DNA hybridized with all five EcoRI/BamHI/HindIII fragments of cloned DNA even at Tm -28 degrees C. Therefore, the causative agent of this PML case was not SV40 but JC virus.

A simple and rapid method for HLA-DQA1 genotyping by polymerase chain reaction-single strand conformation polymorphism and restriction enzyme cleavage analysis.

A simple and rapid method for identification of alleles at the human leucocyte antigen (HLA)-DQA1 locus is described. The polymorphic second exon of the HLA-DQA1 locus was amplified by the polymerase chain reaction (PCR) method. The amplified DNA was analyzed by single-strand conformation polymorphism (SSCP) and restriction enzyme cleavage assay. Using this method, the eight known DQA1 alleles could be distinguished from each other. This paper suggests that the method can be used for quick genotyping of DQA1 alleles, but detecting point mutations at various positions in a fragment as well as new HLA-DQA1 genotypes should also be possible.

Characterisation by restriction endonuclease analysis and plasmid profiling ofVibrio ordaliistrains from salmon(oncorhynchus tshawytschaandoncorhynchus nerka)with vibriosis in new zealand

Abstract Isolates of Vibrio ordalii were recovered from salmon (Oncorhynchus tshawytscha and Oncorhynchus nerka) cultured in New Zealand. The fish suffered from clinical vibriosis, from seven outbreaks in five geographically distinct areas. Affected fish showed gross signs and histopathology similar to those reported from North America. The presence of a plasmid, designated pVO1, approximately 30 kilobases (kb) in size, was seen in all V. ordalii strains examined This plasmid was identical in size and restriction endonuclease cleavage analysis to a plasmid (pMJ101) previously found in V. ordalii strains elsewhere. After togging pVO1 with the transposon Tn1 from the R plasmid RP4, attempts to isolate plasmid‐less derivatives using a variety of curing agents were unsuccessful. All of the New Zealand isolates possessed identical chromosomal DNA cleavage patterns. These patterns were only slightly different from that of the type strain but substantially different from that of the Vibrio anguillarum type strain.

Seroepidemiology of Herpes Simplex Virus and Restriction Endonuclease Cleavage Analysis of Herpes Simplex Virus Type 2 in Okinawa

A seroepidemiologic study of herpes simplex virus (HSV) in Okinawa was performed. A total of 423 serum samples were collected from all over Okinawa, and the positivity rate of antibody against HSV was measured using a passive hemagglutination method. The sero-positive rate for HSV in age groups of over 40 years was 100%. Seven HSV type 2 (HSV 2) isolates were obtained in Okinawa, and DNA preparations from Vero cells infected with the isolates were analyzed using five restriction endonucleases: Bam HI, Hind III, Kpn I, Bgl II and Eco RI. Variations in the genomic region were demonstrated in five of the isolates. Such variations have not been reported previously in HSV 2 in mainland Japan. This is the first report of a seroepidemiologic study of HSV and restriction endonuclease cleavage analysis of HSV 2 in Okinawa, is a subtropical island where HSV is endemic.

Genome analysis of two mosquito species

T m of DNA of Aedes albopictus and Culex bitaeniorhynchus was found to be 78 and 79°C and G-C content 21.2 and 23.6% respectively. High resolution thermal denaturation (HRTD) analysis of both the species shows greater base compositional heterogeneity in GC rich sequences. The highly repetitive DNA sequence accounts for 8–10% of the genome and has a kinetic complexity of 5.1 × 10 5 np. S 1 nuclease resistant repetitive duplexes isolated at Cot 0.1 M.S. from unsheared DNA (>20 kbp) reveal a repeat class of 200–500 bp. Restriction endonuclease cleavage analysis of both the mosquito species show the presence of a family of repeats with MboI and Sau3AI. The digestion of the DNA of both the species with methylation specific restriction enzymes indicates that methylation is extensive and is not restricted to CpG dinucleotides alone. Very significant similarities between the two species were noticed excepting for the HRTD analysis.

Isolation of Multiple Cytomegalovirus Strains from a Patient with Adult T Cell Leukemia

A 66-year-old male with adult T cell leukemia had an ulcer on the left medial thigh. The biopsy of the skin lesion revealed enlarged endothelial cells with acidophilic intranuclear inclusion bodies, suggesting cytomegalovirus (CMV) infection. At autopsy, CMV was isolated from a nodular skin lesion of the scrotum. The urine constantly tested positive for CMV. Restriction endonuclease cleavage analysis of DNA of the isolates from the skin and urine indicated that this patient was infected with two different strains of CMV.

Comparative Restriction Enzyme Cleavage Analysis of Kinetoplast DNA from the Lower Trypanosomatids Isolated in the North-West Region of the USSR

Summary The usefulness of molecular parameters of kinetoplast DNA (kDNA) as taxonomic criteria in the systematics of lower trypanosomatids was studied. 18 previously isolated lower trypanosomatid strains were classified on the basis of their minicircle DNA size and the maxicircle and minicircle cleavage profiles generated with restriction endonucleases. The results of such a classification were compared with the taxonomic status of isolates previously determined with the aid of the traditional systematics criteria. On the basis of cleavage data the isolates were grouped into four distinctly different schizodemes. The first one (A) comprised two Blastocrithidia species, whereas in the second schizodeme (B) three Leptomonas species were contained, thus revealing a good correspondence at the generic level between the 2 types of criteria. The schizodeme D was composed of a single Leptomonas species, which previously was found to differ significantly from the above mentioned three Leptomonas species by the traditional criteria. The most extensive schizodeme C included 12 isolates with a similar though not identical kDNA structure. Previously these isolates were referred to three different genera — Leptomonas, Crithidia and Blastocrithidia . The taxonomic status of several isolates has been determined presumptively. The extent of variability in kDNA structure within this schizodeme was not profound, being scarcely consistent with the different generic status of the isolates. To explain this discrepancy, the different possibilities including unspecific invasions were suggested. The results presented in this study clearly show that the restriction endonuclease analysis of kDNA may be employed as a useful and sensitive tool in developing the lower trypanosomatid taxonomy.

Serological and genetical relationships of three herpesvirus strains from salmonid fish

Serological and genetical relationships of three salmonid herpesviruses [strain H-83, OO-7812 strain of Oncorhynchus masou virus (OMV) and Herpesvirus salmonis] were investigated. Although the serological relationships of these viruses were not distinctly demonstrated by cross-neutralization test, cross enzyme-linked immunosorbent assay revealed that strain H-83 and OMV appeared to be serologically more closely related to each other than to H. salmonis. Strain H-83 was genetically similar to OMV but distinct from H. salmonis by restriction endonuclease cleavage analysis and DNA-DNA hybridization test. These results indicated that strain H-83 should be classified under the same group as OMV, and H. salmonis belong to another group of salmonid herpesviruses.

Immunological and molecular comparison of simian cytomegaloviruses isolated from African green monkey (Cercopithecus aethiops) and Japanese macaque (Macaca fuscata)

Simian cytomegaloviruses (CMV) isolated from African green monkeys and Japanese macaques were compared serologically and genetically. Twenty-three out of 31 sera from Japanese, Taiwan and rhesus macaques were positive for neutralizing antibodies to simian CMVs isolated from both African green monkeys and a Japanese macaque. The neutralizing antibody titers of those sera were slightly higher to the isolates of African green monkeys origin than to that of Japanese macaque origin. Studies with antisera prepared in guinea pigs revealed a partial antigenic cross-reaction between Japanese macaque CMV (JaM-CMV) and African green monkey CMV (AGM-CMV): the antiserum to JaM-CMV neutralized both homologous JaM-CMV and heterologous AGM-CMV, but the antisera to AGM-CMV neutralized only homologous AGM-CMV. DNAs of both AGM-CMV and JaM-CMV were compared by restriction endonuclease cleavage analysis. The individual simian CMV isolates showed unique restriction patterns when cleaved with EcoRI, BamHI, or HindIII. In addition, DNA of JaM-CMV was distinguishable from that of AGM-CMV when cleaved with EcoRI or BamHI.

The isolation and partial sequencing of human bone alkaline phosphatase gene

1. 1. A charon 4A human fetal liver genomic library was screened for human alkaline phosphatase sequences using the cloned human bone cDNA as a hybridization probe. 2. 2. A positive clone was obtained and then characterized by restriction endonuclease cleavage analysis, hybridization experiments and partial DNA sequencing.

Interaction of herpes simplex virus type 2 with a rat glioma cell line.

The interaction between herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and two neural cell lines, mouse neuroblastoma (N1E-115) and rat glioma (C6-BU-1), was investigated. N1E-115 cells were permissive to both types of HSV. In C6-BU-1 cells, on the other hand, all the HSV-1 strains tested so far showed persistent infection, and the infectious virus of HSV-2 strains disappeared spontaneously. The HSV-2-infected C6-BU-1 cells were positive for HSV-2-specific DNA sequences, virus-specific RNA, HSV-2-specific antigens and thymidine kinase activity, when no infectious virus was detected. The HSV-2 was reactivated from those C6-BU-1 cells by superinfection with murine cytomegalovirus (MCMV), but not with UV-irradiated MCMV or human cytomegalovirus. The reactivated HSV-2 was identical to the parental virus, when examined by restriction endonuclease cleavage analysis.

DNA sequence of Crithidia fasciculata kinetoplast minicircles

Kinetoplast DNA (kDNA) networks of the insect trypanosomatid Crithidia fasciculata strain CF-C1 contain a nearly homogeneous population of kDNA minicircles as judged by restriction enzyme cleavage analysis. We have determined the entire nucleotide sequence of the major class of minicircles by analyzing M13 phage clones carrying half-length segments of the kDNA minicircle molecules. The 12 nucleotide sequence d(G-G-G-G-T-T-G-G-T-G-T-A) is the longest sequence common to kDNA minicircles from several trypanosome species examined to date. Two copies of this universal minicircle sequence were identified 180° apart as direct repeats within the C. fasciculata kDNA minicircles. In addition, these universal minicircle sequences are contained within direct repeats with nearly identical sequences of 173 and 177 base pairs (bp) in length. These sequences are also conserved in the same arrangement in minor sequence classes of minicircles from this strain. Site-specific discontinuities on both strands of the minicircle, identified previously in minicircle replication intermediates, were localized within the 173 and 177 bp conserved sequences. These sequences were also found to have extensive homology with similar conserved sequences in kDNA minicircles from Leishmania tarentolae. We suggest that the two conserved sequences, each containing a single copy of the universal minicircle sequence, represent replication origins in the Crithidia minicircles.