Restriction Endonuclease Patterns Research Articles

Childhood Acute Bacterial Meningitis in the Suda: An Epidemiological, Clinical and Laboratory Study

The aims of the present study were to document the epidemiology, clinical features and complications of childhood acute bacterial meningitis (ABM) in The Sudan during both an inter-epidemic (endemic) period (1985-1986), and the 1988 serogroup A epidemic; and to examine the phenotypic and genetic similarities and differences of Neisseria meningitidis strains isolated in The Sudan and Sweden. A new enzyme immunoassay test (Pharmacia Meningitis EIA-Test) was evaluated as a potential rapid diagnostic method for the detection of Haemophilus influenzae (HI) type b, Neisseria meningitidis (MC) and Streptococcus pneumoniae (PNC). The test was found to have good sensitivity (0.86) and specificity (0.95) in the inter-epidemic period; and to be adaptable to the field work in The Sudan during the 1988 MC epidemic. During inter-epidemic (endemic) situations in The Sudan, greater than 90% of childhood ABM was caused by one of the three organisms, HI type b, MC and PNC. HI accounted for 57% of the cases. The peak incidence (76%) of HI cases was in infants (less than 12 months) similar to the situation in other African countries. The overall case fatality ratio was 18.6%. Prospective follow-up of survivors for 3-4 years revealed that an additional 43% either died or had permanent neurological complications, the most prevalent and persistent of which was sensorineural hearing loss recorded in 22% of long term survivors. Post-meningitic children were found to have significantly lower intelligence quotients (92.3 +/- 13.9) than their sibling controls (100.7 +/- 10.2, P = 0.029). Features of the large serogroup A sulphonamide resistant MC epidemic (February-August 1988) in Khartoum are described. An estimated annual incidence of 1,679/100,000 was recorded at the peak of the epidemic. The highest attack rate was in young children less than 5 years, as in many other African countries; nevertheless, a high morbidity was observed in adults (31% of the cases greater than or equal to 20 years). The clinical features, mortality (6.3%) and short term sequelae in Sudanese children were generally within the framework described for MC disease elsewhere. Detailed analysis of MC isolates from Sudan and Sweden by characterizing their electrophoretic enzyme types, DNA restriction endonuclease pattern and outer membrane proteins, revealed that serogroup A MC clone III-1 was responsible of The Sudan epidemic in 1988 and has been the dominant serogroup A organism in Sweden since 1973. The Sudanese strains isolated prior to the epidemic (1985) were clone IV-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondrial DNA Restriction Endonuclease Patterns of Sorghums at Various Ploidy Levels

Mitochondrial DNA from Sorghum versicolor (2n=10), Sorghum bicolor race bicolor (2n=20), and Sorghum halepense (2n=40) was digested with an endonuclease

The human α-amylase multigene family consists of haplotypes with variable numbers of genes

Polymorphic amylase protein patterns have suggested the presence in the human genome of various haplotypes encoding these allozymes. To investigate the genomic organization of the human α-amylase genes, we isolated the pertinent genes from a cosmid library constructed of DNA from an individual expressing three different salivary amylase allozymes. From the restriction maps of the overlapping cosmids and a comparison of these maps with the restriction enzyme patterns of DNA from the donor and family members, we were able to identify two haplotypes consisting of very different numbers of salivary amylase genes. The short haplotype contains two pancreatic genes ( AMY2A and AMY2B) and one salivary amylase gene ( AMY1C), arranged in the order 2B-2A-1C, encompassing a total length of approximately 100 kb. The long haplotype spans about 300 kb and contains six additional genes arranged in two repeats, each one consisting of two salivary amylase genes ( AMY1A and AMY1B) and a pseudogene lacking the first three exons ( AMYP1). The order of the amylase genes within the repeat is 1A-1B-P1. All genes are in a head-to-tail orientation except AMY1B, which has the reverse orientation with respect to the other genes. Analysis of somatic cell hybrids confirmed the presence of these short and long haplotypes. Furthermore, we present evidence for the existence of additional haplotypes in the human population and propose a general model for the evolution of the human α-amylase multigene family. A general designation 2B-2A-(1A-1B-P) n-1C can describe these haplotypes, n being 0 and 2 for the short and the long haplotypes presented in this paper, respectively.

Classification of virulent bacteriophages of Streptococcus salivarius subsp. thermophilus isolated from yoghurt and Swiss-type cheese

Twelve isometric-headed bacteriophages virulent against Streptococcus salivarius subsp. thermophilus were differentiated into three subgroups by analysis of the phage genomes and the structural proteins. Subgroup I is composed of two phages (P6 and P8) with a genome size of 41.2 and 44.2 kb pairs, respectively, complete DNA homology, and identical protein composition (main proteins of sizes 39.8, 24.0, 14.8 kilodaltons in sodium dodecyl sulphate-polyacrylamide gel electrophoresis). One phage (a10/J9) with low DNA homology to the other phages was classified into subgroup II. Subgroup III consists of nine phages with a genome size of 33.8 to 36.7 kb pairs and two major structural proteins (30.9 and 24.0 kilodaltons, or 30.9 and 26.3 kilodaltons). In general, phages with different host spectra revealed different restriction enzyme patterns, and DNA homologies of various degrees were detected among all phages tested.

Characteristics and comparative study of fiveRhizobium meliloti bacteriophages

FiveRhizobium meliloti bacteriophages isolated from soil or lysogenic bacteria and belonging to Bradley's group B or theSiphoviridae family of tailed phaes were studied. They are of identical morphology, showing isometric heads and long, noncontractile tails with transverse bars. They are temperate and closely related by host range, DNA restriction endonuclease patterns and homology, DNA mass, serological properties, adsorption velocity, and latent period. However, the phages can be divided into three groups on the basis of burst size and frequency of lysogenization.

Analysis of chloroplast and mitochondrial DNAs in asymmetric somatic hybrids between tobacco and carrot

Chloroplast and mitochondrial DNAs have been examined by comparison of restriction enzyme patterns in asymmetric hybrid plants, resulting from the fusion between leaf mesophyll protoplasts of Nicotiana tabacum (Solanaceae), and irradiated cell culture protoplasts of Daucus carota (Umbellifereae). These somatic hybrids with normal tobacco morphology were selected as a consequence of the transfer of methotrexate and 5-methyltryptophan resistance from carrot to tobacco. The restriction patterns of chloroplast DNAs in somatic hybrids were indistinguishable from the tobacco parent. However, we found somatic hybrids with mitochondrial DNA significantly different from either parent, as judged by analysis of fragment distribution after restriction enzyme digestion. The possible formation of altered mitochondrial DNA molecules as the result of parasexual hybrid production between two phylogenetically highly divergent plant species will be discussed.

Genetic heterogeneity amongFrankiaisolates from root nodules of individual actinorhizal plants

Genetic variations among selected Frankia isolates from nitrogen-fixing root nodules harvested from an individual actinorhizal plant (Elaeagnus angustifolia L. or Shepherdia argentea Nutt.) were estimated by restriction fragment analysis of their total genomic DNA. The presence of plasmids and their restriction enzyme patterns were used as additional criteria. Certain isolates from separate nodules on the same plant were found indistinguishable, being probably clones of the same strain. An endophytic passage of a strain isolated from S. argentea on another host plant, Hippophae rhamnoides L., did not modify the structural characteristics of the genome in the reisolates obtained. However, in some cases, especially when restriction endonucleases cleaving Frankia DNA into relatively small fragments were used, multiple infection of the actinorhizal plants with different Frankia strains and the presence of more than one strain in a nodule were demonstrated. Some aspects of variability in natural populations of Frankia are discussed.

Restriction endonuclease analysis of mitochondrial DNA from sorghum with fertile and male-sterile cytoplasms

Mitochondrial DNA from four paired (fertile and male-sterile) lines and six isocytoplasmic strains of sorghum (Sorghum bicolor (L.) Moench) were fragmented by endonucleases and their electrophoretic patterns were examined. Cytoplasmic male sterile lines differed from their male-fertile counterparts consistently. Among the isocytoplasmic strains, KS 36A (S. verticilli-florum cytoplasm), KS 38A (S. conspicum cytoplasm), and KS 39A (S. niloticum cytoplasm) showed minor differences from the other strains. Results suggest that restriction endonuclease patterns are useful in detecting differences in mitochondrial genomes.

Increased level of mitochondrial gene expression in polyps of familial polyposis coli patients

To elucidate the mechanism of polyp formation in familial polyposis coli patients, cDNA clones, expressed differently between normal colonic mucosae and polyps, were screened. Three polyp-derived cDNA clones showed increased levels of transcription under neoplastic conditions. Among them, clone P6 was determined to be derived from the mtDNA encoding parts of subunit 1 of NADH dehydrogenase and 16S rRNA. The number of copies and restriction enzyme patterns of the mtDNA did not vary in either normal or neoplastic tissues, and a level of different mitochondrial mRNA was increased in the same proportion, suggesting a change in the rate of synthesis and/or degradation of the mtRNA. No cDNA clone which was expressed higher in normal colonic mucosae than in polyps was isolated from the cDNA library derived from normal mucosae.

Inactivation of Nonoccluded and Occluded Baculoviruses and Baculovirus-DNA Exposed to Simulated Sunlight

Natural sunlight is the most significant environmental factor affecting the stability of microbial insecticides formulated from occluded entomopathogenic viruses. Theoretically the crystalline protein matrix that occludes and surrounds each virion containing DNA should provide some protection against inactivation by sunlight-UV. No significant differences, however, were obtained in either the extent or rate of inactivation when nonoccluded, polyhedra derived virions (PDV) and tissue culture derived virions (TCNOV), or virions occluded in polyhedral inclusion bodies (PIBOV) of the Singly-enveloped nuclear polyhedrosis virus of Heliothis species (HzSNPV) were exposed to a simulated sunlight-UV source (SUV). The residual half-life of PDV, TCNOV, and PIBOV, as measured by bioassay against susceptible larvae of Heliothis virescens , was 6.3, 6.3, and 5.8 h, respectively. There was smearing of the EcoRI restriction enzyme (REN) patterns and an increase in lower molecular-weight species after exposure to SUV of both naked viral DNA and DNA of virions occluded in polyhedral inclusion bodies (PIB). Production of higher molecular-weight species of DNA, however, only was detected in DNA from PIBOV exposed to SUV. Changes in REN patterns of viral DNA were never detected with shielded-exposed HzSNPV.

Abnormal glucocorticoid receptor gene and mRNA in primary cortisol resistance

Abnormal steroid hormone receptors have been implicated as causing several forms of primary steroid hormone resistance in humans, but as yet no abnormality has been described at the gene level. We describe the analysis of the mRNA and genomic DNA from the Epstein-Barr (EB) virus transformed cells of two siblings with Primary Cortisol Resistance. The cells of the propositus and his brother show a decreased level of glucocorticoid receptor (GR) mRNA, and the genomic DNA of both individuals shows an altered restriction enzyme pattern with the restriction enzyme Bgl II, one of eleven restriction enzymes tested. The genomic differences could be detected with a probe specific for the putative steroid binding domain of the human GR gene.

Correction of chlorophyll-defective male-sterile winter oilseed rape (Brassica napus) through organelle exchange: molecular analysis of the cytoplasm of parental lines and corrected progeny

Cytoplasmic differences between male-fertile and male-sterile Brassica napus as well as Raphanus sativus were investigated. Plastids of the male-fertile B. napus were found to differ from those of male-sterile B. napus and R. sativus with respect to DNA restriction enzyme patterns. Differences between male-fertile and male-sterile B. napus mitochondria were detected not only in the restriction fragment patterns of their DNA, but also at the level of expression by in organello translation of mitochondrial polypeptides.The chlorophyll deficiency obtained upon transferral of the male-sterility-conferring radish cytoplasm to a winter variety of B. napus had been corrected earlier through protoplast fusion. The cytoplasmic composition of the corrected lines was analysed using DNA restriction analysis and in organello translation. The stability of the recombined cytoplasm in the corrected lines was confirmed by analysis of the subsequent seed-derived generation.

Genome analysis of adenovirus 4a, a causative agent of pharyngoconjunctival fever and respiratory diseases in Brazil.

An outbreak of pharyngoconjunctival fever caused by adenovirus (Ad) occurred in 1976 in Belem (northern Brazil). By restriction endonuclease analysis, it was shown that the type responsible was Ad4a, a variant of Ad4 prototype (Ad4p) and previously described as causative agent of ocular diseases. In an epidemiologic study carried out in Rio de Janeiro and Belem between 1977 and 1986, 148 Ads were isolated from children with respiratory disease. All five Ad4 strains encountered during this study were also Ad4a. This type, circulating in South America, can therefore cause both ocular and respiratory diseases. No Ad4p was isolated during this study. A percent of comigrating restriction fragments of only 50% between Ad4p and Ad4a was calculated from 13 restriction enzyme patterns. The restriction maps of Ad4a for nine endonucleases are presented and compared with those of Ad4p.

PM2 bacteriophage plaque morphology mutants exhibit differences in DNA length and restriction endonuclease patterns

A large plaque (LP) and a small plaque (SP) variant of PM2 bacteriophage were isolated from a mixture of the two plaque variants and were grown separately in the appropriate host bacterium,Alteromonas espejiana. They have remained pure for approximately one year from the original isolation. Restriction endonuclease analyses revealed differences in theHaeIII restriction profile between the two variants.HaeIII fragment 1 of the SP DNA was found to be smaller than the corresponding fragment from the LP variant DNA, whereas fragment 7 from the SP DNA was slightly larger than the same fragment from LP DNA. Electron microscopy of heteroduplexes formed between the DNAs from the two variants revealed that the deletion in fragment 1 mapped very close to the junction betweenHaeIII fragments 1 and 13 on the physical map of PM2 DNA. The difference in DNA length between the two variants results from addition or deletion mutations.

Molecular analysis of organelle DNA of different subspecies of rice and the genomic stability of mtDNA in tissue cultured cells of rice

Chloroplast (ct) and mitochondrial (mt) DNAs were isolated from two subspecies of rice (Oryza sativa), japonica (Calrose 76) and indica (PI353705) and compared by restriction endonuclease fragment pattern analysis. Similarly, PI353705 (A5) mtDNA was also compared with the mtDNA of its long term tissue cultured line, BL2. Variation in the ctDNA of the 2 subspecies was detected with two (AvaI and BglI) of the 11 restriction endonucleases tested, whereas their mtDNAs showed considerable variation when restricted by PstI, BamHI, HindIII and XhoI endonucleases. Thus, the chloroplast DNA was more highly conserved than the mtDNA in the subspecies comparisons. Only minor variation was observed between the restriction endonuclease patterns of the mtDNAs of BL2 and A5. Southern blots of mtDNA were hybridized with heterologous probes from maize and spinach organelle genes. Differences were found in the hybridization patterns of the two subspecies for six of the eight (mitochondrial and chloroplast) probes tested. Two of the seven (mitochondrial) probes (coxII and 26S rRNA) detected tissue culture generated variation in mtDNA. The relative values of restriction endonuclease and hybridization patterns for studying phylogenetic and genetic relationships in rice are discussed.

Acid phosphatases of Escherichia coli: molecular cloning and analysis of agp, the structural gene for a periplasmic acid glucose phosphatase.

Several unknown Escherichia coli genes for different species of acid phosphatase were cloned in vivo with the plasmid Mu dII4042. When present in a multicopy state, each gene promoted hydrolysis of p-nitrophenyl-phosphate at acidic pH. Among seven recombinant clones that encoded periplasmic acid phosphatase activities, five different genes could be distinguished by the pH optimum and substrate preference for the enzyme and by the restriction enzyme pattern. A 1.7-kilobase recombinant DNA fragment, common to two clones, was inserted into plasmid pBR322 and shown to contain a new gene, agp, which leads to the overexpression of the periplasmic acid glucose-1-phosphatase, a dimer of a 44-kilodalton polypeptide. Fusions of agp to gene phoA deprived of its own signal sequence conferred an alkaline phosphatase-positive phenotype to bacteria, showing the presence of an export signal on agp. The resulting hybrid proteins were characterized by immunoprecipitation with an antiserum directed against purified acid phosphatase or against alkaline phosphatase, showing that agp is the structural gene of the acid phosphatase. The beginning, the orientation, and the end of gene agp on the cloned DNA fragment were determined by the characteristics of such hybrid proteins.

Plasmids in Vibrio salmonicida isolated from salmonids with hemorrhagic syndrome (Hitra disease).

Vibrio-like isolates from Atlantic salmon (Salmo salar Linnaeas) and a few from rainbow trout (S. gairdneri Richardson) suffering from hemorrhagic syndrome (Hitra disease), also called cold-water vibriosis, a disease of great importance in Norwegian fish farming, were examined for plasmid content. Of 84 strains isolated from 1982 to 1984, 70 (83.3%) had a common 21-megadalton (MDa) plasmid. A 3.4-MDa plasmid was found in 58 of the strains with the 21-MDa plasmid, and a 2.8-MDa plasmid was found in 23 of the strains with both the 21- and 3.4-MDa plasmids. The strains were isolated from fish farms along the western and northern coasts of Norway. Ten (11.9%) of the strains possessed a 61-MDa plasmid in addition to a 21-MDa plasmid. Two strains had only a 21-MDa plasmid. Of the 84-Vibrio-like isolates, 14 did not harbor plasmids identical in mass to any other plasmids found in this material. Vibrio salmonicida strains, 257 in all, isolated from salmonids with the same disease from the same area from July 1986 to July 1987, all possessed a 21-MDa plasmid, either alone or in addition to a 3.4-MDa plasmid, or a combination of 3.4- and 2.8-MDa plasmids. Six of the strains had a 5.5-MDa plasmid instead of the 3.4-MDa plasmid. The restriction endonuclease patterns of the plasmids of similar molecular mass reflected similar nucleotide sequences. The plasmid content detected in isolates of V. salmonicida obtained from a coastline of more than 2,000 km and over a period of almost 6 years is stable.

Selection of nuclear polyhedrosis viruses as biological control agents ofSpodoptera exigua [Lep.: Noctuidae

The virulence of 5 nuclear polyhedrosis viruses infectious for larvae of beet armyworm,Spodoptera exigua, was studied and their potential as biological control agents of this accidentally introduced pest in Dutch greenhouse crops is discussed. Three of the virus isolates were collected from deceased beet armyworm larvae found in Dutch greenhouses. Based on restriction endonuclease patterns of their DNA they appeared to be closely related toMamestra brassicae nuclear polyhedrosis virus (MbMNPV) and therefore were named MbMNPV-NL80, MbMNPV-NL82 and MbMNPV-NL83. These isolates were not related toAutographa californica MNPV (AcMNPV) or toSpodoptera exigua MNPV (SeMNPV), both originating from the USA.

Molecular evolution of the mouse proline-rich protein multigene family. Insertion of a long interspersed repeated DNA element.

Proline-rich proteins (PRPs) in the salivary glands of mice, rats, and hamsters are encoded by tissue-specific inducible multigene families. Mouse PRP genes are located on chromosome 8, and transcription is dramatically induced (about 70-fold) by isoproterenol treatment. Clones containing two nonallelic PRP genes (MP2 and M14) were isolated from cosmid and phage libraries of CD-1 mouse genomic DNA. The cloned regions comprise a contiguous block of 77 kilobase pairs of the mouse genome. Restriction mapping established the physical lineage of PRP genes MP2 and M14, and they are tandemly arrayed. The DNA sequence analysis presented in this report suggests that genes M14 and MP2 (Ann, D. K., and Carlson, D. M. (1985) J. Biol. Chem. 260, 15863-15872) arose via a gene duplication of a common ancestor. Two major differences between M14 and MP2 were observed. PRP gene MP2 has 13 tandemly arrayed 42-nucleotide repeats in exon II, whereas M14 has 17 repeats, and PRP gene M14 has an insertion by transposition of a 2-kilobase pair member of the long interspersed repeated DNA (LINE) family (LIMd) into intron I. The evolution of this PRP multigene family has been dominated by intra-exonic amplification of repeating nucleotide units coding for these and other proline-rich repeated peptides and by gene duplication. The LIMd element gives rise to heterogenous EcoRI, BamHI, and HindIII restriction enzyme patterns, and this insertion is also present in BALB/c, C57BL/6J, and DBA/2J mice.

Characterization of two DNA clones specific for identification of Corynebacterium sepedonicum

Escherichia coli TB1 was transformed with pUC9 containing fragmented DNA (4–10 kilobases (kb)) from Corynebacterium sepedonicum. The resulting genomic bank was screened by a dot blot assay to identify clones specifically hydridizing to C. sepedonicum DNA and not to the DNA of several other Gram-positive and Gram-negative bacteria. Two clones (III24 and III31) were selected because of their ability to strongly hybridize to C. sepedonicum DNA and weakly hybridize to the DNA of C. michiganense, Erwinia carotovora, Agrobacterium tumefaciens, Bacillus subtilis, Pseudomonas solanacearum, Micrococcus luteus, and Arthrobacter globiformis. These two clones were also specific for C. sepedonicum DNA when tested against the DNA from 30 isolates of soil bacteria. Restriction enzyme analysis has shown that the two clones have an insert of 8 kb (III24) and 4 kb (III31). On the basis of restriction enzyme patterns, one clone (III24) does not correspond to plasmid pCL 50, a cryptic plasmid found in several C. sepedonicum isolates. Because purified III24 and III31 DNA can be used to detect approximately 1 ng of C. sepedonicum genomic DNA, the two clones can complement serological or biological detection methods. This could be useful, especially when a high degree of specificity is required for detection or identification of this plant pathogen.