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K Umene

Publications and source records attributed to K Umene.

At least 55 records · Page 3Linked to original sources

[Simultaneous in situ detection of viral antigens and RNA in brain tissues from a patient with herpes simplex encephalitis by immunocytochemistry and in situ hybridization].

In order to elucidate the correlation between herpes simplex virus (HSV-1) and the central nervous system tissue, we performed the simultaneous detection of viral antigens and RNA in the brain tissue sections from a patient with herpes simplex virus (HSV) encephalitis using immunocytochemistry and in situ hybridization. In the present study the hybridization protocol reported by Brahic M et al. in 1984 were applied for the simultaneous detection of viral RNA and antigens with a few modification. The sections were first immunocytochemically stained to detect HSV-1 antigens by ABC method, and then hybridized with 3H-labelled HSV-1 cDNA probe for the detection of RNA after the acetylation of slides for the prevention of nonspecific bindings of isotope to slides. In the present study, viral antigens were immunocytochemically stained to brown-colored deposits located in the cytoplasm and nucleus whereas viral RNA were detected as the accumulation of many silver grains over the nuclei or cytoplasm. In this case the light microscopic findings in a part of temporal lobe showed multiple areas of necrosis mainly involving the gray matter and a few inflammatory changes such as perivascular cell cuffings. HSV-1 infected Vero cells as positive control demonstrated both antigens and RNA as shown in Fig.1 a. However, no hybridization signals and color deposits were observed in uninfected Vero cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Viral↗

Hematopoietic growth factors (BPA and Epo) induce the expressions of c-myc and c-fos proto-oncogenes in normal human erythroid progenitors.

We investigated serial expressions of eight proto-oncogenes during in-vitro differentiation of normal human burst-forming unit, erythroid (BFU-E), and found that c-myc and c-fos are expressed in progenies of BFU-E. The expressions of the two proto-oncogenes correlated to the replating efficiency and adversely to erythroid differentiation. The absence of hematopoietic growth factors decreased the expressions, but the addition of erythropoietin together with burst promoting activity induced a re-expression of the c-myc and c-fos after 2 h of incubation. These observations suggest that the c-myc and c-fos proto-oncogenes have a physiological role in the proliferation of erythroid progenitors and that activations of the two proto-oncogenes are early cellular events after the stimulation by hematopoietic growth factors.

Cell Differentiation↗

Restriction endonucleases recognizing DNA sequences of four base pairs facilitate differentiation of herpes simplex virus type 1 strains.

Variation of restriction endonuclease cleavage patterns between herpes simplex virus type 1 (HSV-1) strains was investigated using restriction endonucleases recognizing DNA sequences of four base pairs (4-bp enzymes) (HaeIII, HhaI, Sau3AI). The analysis made feasible identification of variations not heretofore detectable, using enzymes recognizing DNA sequences of six base pairs (BamHI, KpnI, SalI), and proved to be most useful for distinction and classification of HSV-1 strains. Ten of the fifteen strains analysed using 4-bp enzymes were closely related, and were put into group 1, and the other five not so related strains were classed into the non-group 1. The observations suggested a possible common ancestor from which group 1 strains had derived.

Animals↗

Transition from a heterozygous to a homozygous state of a pair of loci in the inverted repeat sequences of the L component of the herpes simplex virus type 1 genome.

The behavior of herpes simplex virus type 1 heterozygous isolates, in which the two inverted repeats of the L component (RL) were differentiated by a polymorphism marker (the presence [type B] or absence [type A] of a SalI site), was investigated. The progeny viruses derived from the heterozygote (A/B) consisted of heterozygotes (A/B), type A homozygotes (A/A), and type B homozygotes (B/B). The heterology between RL, albeit tolerated, was unstable, as is the case with heterology between the repeats of the S component. The two repeats TRL (terminal) and IRL (internal) were equipotent in generating homozygotes from a heterozygote. Data obtained from an analysis of 426 progeny viruses derived from heterozygous clones supported the hypothesis that the two loci in RL of a herpes simplex virus type 1 genome are determined as a random combination of the corresponding two loci in RL of the parent virus and that the ratio of heterozygotes/type A homozygotes/type B homozygotes in the progeny viruses from a heterozygote is expected to be 2:1:1. An ephemeral dominance of one type of homozygote over the other was observed in subclones from several heterozygous clones.

Animals↗

Expression of c-myc oncogene during differentiation of human burst-forming unit, erythroid (BFU-E).

We investigated the serial expression of c-myc oncogene during differentiation of normal human burst-forming unit, erythroid (BFU-E), in vitro, together with the replating efficiency and the expression of transferrin receptors. We found that the c-myc oncogene was expressed in the early progeny of BFU-E and that this expression decreased during erythroid differentiation. This change of the c-myc expression was well correlated to changes in the replating efficiency and the expression of transferrin receptors. These observations indicate that the c-myc oncogene probably has a physiological role in normal human hematopoiesis as well as leukemic hematopoiesis.

Cell Differentiation↗

Conversion of a fraction of the unique sequence to part of the inverted repeats in the S component of the herpes simplex virus type 1 genome.

A novel genome variant of herpes simplex virus type 1 (HSV-1) was isolated, in the S component of which a fraction of the unique sequence (map units 0.865 to 0.880) was converted to part of the diploid inverted repeats and another fraction of the unique sequence (map units 0.937 to 0.955) had been deleted. The S component of the variant consisted of a shortened unique sequence (map units 0.880 to 0.937) and a pair of elongated inverted repeats (map units 0.820 to 0.880, and 0.937 to 1.000). The conversion occurred as a result of a recombination event between two points (map units 0.880 and 0.937) having a 5 base pair stretch (5'-CCCCG-3') of homology, in an inverted direction in the unique sequence. An involvement of the mechanism causing L-S inversion was inferred to account for the generation of the variant, as there are multiple copies of the 5'-CCCCG-3' stretch in the 'a' sequences. The occurrence of the variant indicates that the products of HSV-1 genes US9, US10, US11 and US12 are unnecessary for an HSV-1 productive infection in tissue culture cells, and also suggests the presence of a mechanism whereby expansion of the inverted repeats could occur.

Base Sequence↗

Intermolecular recombination of the herpes simplex virus type 1 genome analysed using two strains differing in restriction enzyme cleavage sites.

Intermolecular recombination of herpes simplex virus type 1 (HSV-1) was studied by analysing the segregation of strain-specific restriction enzyme cleavage sites among progeny viruses produced after co-infection by two HSV-1 strains differing in eight restriction enzyme cleavage sites. Out of 93 progeny viruses examined, 51 clones were recombinant, and crossover sites of the recombinants were mapped on the HSV-1 genome. These sites were distributed evenly in the unique sequence of the L component (UL) and the recombination frequency in UL was estimated to be 1.12 per genome length, or 0.007 per kilobase pair. No evidence was obtained to support the existence of enhanced intermolecular recombination events in the regions containing inverted repeats and the L-S junction in comparison with the recombination frequency in UL. The finding of recombinants in an arrangement that minimized the number of crossover events suggested the participation of both of two arrangements of the L component of parental DNA (P or IS, and IL or ISL) in the generation of the recombinants. The possibility of a preference for P or IS over IL or ISL arrangements remains to be determined.

Chromosome Mapping↗

Isolation of novel herpes simplex virus type 1 derivatives with tandem duplications of DNA sequences encoding immediate-early mRNA-5 and an origin of replication.

Two naturally occurring variations of herpes simplex virus type 1 (Patton strain) with novel tandem DNA sequence duplications in the S component were isolated, and the DNA was characterized. These variants were identified among a number of plaque isolates by the appearance of new restriction enzyme fragments that hybridized with radiolabeled DNA from the BamHI Z fragment (map coordinates 0.936 to 0.949) located in the unique S region. One isolate, SP26-3, carried a 3.1-kilobase-pair duplication defined by recombination between a site in the BamHI Z fragment and a site near the origin of replication in the inverted repeat sequence of the S component carried by the EcoRI H fragment. The other isolate, SP22-4, carried a 3.5-kilobase-pair duplication defined by a recombination event between a tandem repeat array in the BamHI Z fragment and a site near the amino terminus of the Vmw175 gene in the S-region inverted repeat sequence contained in the EcoRI K fragment. Both duplicated segments contained the entire immediate early mRNA-5 coding region as well as the origin of replication located in the inverted repeat sequence of the S component. The DNA sequence of each duplication joint was determined.

Base Sequence↗

Variability of the region of the herpes simplex virus type 1 genome yielding defective DNA: SmaI fragment polymorphism.

A SmaI map of class I defective DNA of herpes simplex virus type 1 (HSV1) was constructed using a set of deletion hybrid phages. Four SmaI fragments on the defective DNA had a variability in length common among 15 HSV1 isolates: the 1.45 kilobase (kb) fragment located within the BamHI-Z (map coordinates 0.936-0.949) fragment, the 0.92-kb fragment neighboring on the 'a' sequence, the 0.44-kb fragment containing the intervening sequence of immediate-early mRNA-5 gene, and the 0.205-kb fragment corresponding to the 'a' sequence. The four SmaI fragments have several sets of reiterated sequences, among which the 1.45- and 0.92-kb fragments hybridized with mammalian cellular DNAs (human, monkey, rabbit, and mouse).

DNA Restriction Enzymes↗

Herpes simplex virus type 1 restriction fragment polymorphism determined using southern hybridization.

Regions of herpes simplex virus type 1 (HSV-1) DNA with variation in the size of restriction endonuclease fragments were identified by comparison of the BamHI, KpnI or SalI restriction endonuclease digestion patterns among 15 HSV-1 isolates after hybridization with specific 32P-labeled cloned HSV-1 DNA fragments. Of the types of restriction fragment polymorphism identified, one was a strain with a distinctly different restriction fragment than the prototype (loss or gain of restriction sites). Another type, the specific fragment varied only in size among strains. Thirteen distinct variations were identified. Ten were mapped to the unique sequence of the L component; two to the inverted repeat of the L component and one to the inverted repeat of the S component. The presence of a common ancestor from which some isolates of HSV-1 might derive was deduced from an analysis of the distribution of the thirteen variations among the 15 HSV-1 isolates.

Base Sequence↗

Tandem repeated DNA in an intergenic region of herpes simplex virus type 1 (Patton).

When the entire US region of HSV-1 (Patton) was cloned as an EcoRI fragment in bacteriophage lambda gtWES, the BamHI B6B5 fragment was observed to vary in size among independent isolates [Umene and Enquist, Gene 13 (1981) 251-268]. This fragment polymorphism also occurred in DNA of HSV-1 single plaque isolates. We report here that this heterogeneity is due to variation in copy number of a 15-bp tandem repeat of sequence 5'-CCACTCCCCACCCAC-3', which apparently lies in an intergenic region of the HSV-1 DNA.

Base Sequence↗

Isolation of human DNAs homologous to the BamHI-Z fragment of herpes simplex virus type 1 DNA.

Human DNA hybridizes with the BamHI-Z fragment (map coordinates 0.936 to 0.949) of Herpes simplex virus type 1 (HSV-1) DNA. To characterize the BamHI-Z homologue of human DNA, we isolated six independent hybrid phages with a sequence homologous to the BamHI-Z fragment from a human genomic DNA library. Three of the six had a common 1.2-kb BamHI-EcoRI fragment homologous to the BamHI-Z, and this fragment existed as 10-60 copies per human haploid genome. A 0.29-kb MboII segment of the BamHI-Z fragment was found to be responsible for the homology with the 1.2-kb BamHI-EcoRI fragment.

Cloning, Molecular↗

Reiterated sequences within the intron of an immediate-early gene of herpes simplex virus type 1.

We describe the nucleotide sequence of a herpes simplex virus type 1 DNA fragment containing the intron of the immediate-early mRNA-5 (IE mRNA-5) gene. The location of the intron within this fragment was determined by a Berk & Sharp nuclease S1 protection analysis, and by cloning and sequencing cDNA containing sequences overlapping t he IE mRNA-5 splice point. We found that the 149 base pair (bp) intron contained four copies of an identical 23 bp GC rich tandem repeat followed by a further reiteration consisting of the first 15 bp only.

Cloning, Molecular↗

A deletion analysis of hybrid phage carrying the US region of Herpes simplex virus type 1 (Patton). I. Isolation of deletion derivatives and identification of chi-likes sequences.

The EcoRI-H fragment (15.4 kb) of Herpes simplex virus type 1 (HSV-1) has been cloned in lambda gtWES in both orientations. This fragment contains the entire US region and has about 900 bp of terminal redundant sequences derived from the internal and terminal repeats of the S region. 56 independent plaque-forming deletion derivatives of the lambda gt/WES::EcoRI-H hybrid phage were isolated using either EDTA resistance or ability to grow on Escherichia coli(P2) lysogens as selective methods. The endpoints of these deletions were located using nine restriction enzymes that cleave within the EcoRI-H fragment. All of the deletions have at least one endpoint within the cloned fragment. Several unusual features of the lambda hybrids, including heterogeneity of a particular region in the HSV-1 EcoRI-H fragment and the presence of chi-like sequences in the US region of HSV-1, are discussed.

Bacteriophage lambda↗

A deletion analysis of lambda hybrid phage carrying the US region of Herpes virus type 1 (Patton). II. Construction of an SmaI map.

The 15.4 kb EcoRI-H fragment of Herpes simplex virus type 1 (HSV-1) strain Patton, which contains the entire short unique (US) region, has been cloned in bacteriophage lambda. The fragment contains a terminal redundancy of about 900 bp that represents the S region terminal-repeat sequences. The restriction enzyme SmaI cleaves the EcoRI-H fragment at more than 30 sites. We have constructed an SmaI map of this fragment using thirteen isolates of lambda gtWES hybrid bacteriophage that carry various deletions of the EcoRI-H fragment.

Bacteriophage lambda↗

Lambda bacteriophage-mediated transduction of ColE1 deoxyribonucleic acid having a lambda bacteriophage-cohesive end site: selection of packageable-length deoxyribonucleic acid.

An in vitro recombinant ColE1-cos lambda deoxyribonucleic acid (DNA) molecule, pKY96, has 70% of the length of lambda phage DNA. The process of lambda phage-mediated transduction of pKY96 generated a small amount of transducing phage particles containing ColE1-cos lambda DNA molecules of 80 or 101% of the length of lambda phage DNA, in addition to those containing original pKY96 DNA molecules. The newly isolated larger plasmid DNAs were transduced 100 times more efficiently than pKY96 DNA. Their structures were compared with that of a prototype pKY96 DNA, and the mechanism of the formation of these molecules is discussed.

Bacteriophage lambda↗

Packaging of ColE1 DNA having a lambda phage cohesive end site.

The mechanism of lambda phage-mediated transduction of hybrid colicin E1 DNAs of various lengths was studied, and factors influencing the formation of these transducing particles were investigated. The results were as follows: 1. The presence of a cohesive end site of lambda phage (coslambda) on colicin E1 DNA was essential for packaging of the DNA. 2. Packaging of colicin E1 DNAs, which carry coslambda with molecular sizes corresponding to 68% of that of lambda phage DNA, was observed in the absence of all known recombination functions of E. coli K-12 and of lambda phage. 3. Hybrid colicin E1 DNAs having coslambda with molecular sizes corresponding to 28% of that of lambda phage DNA were packaged within lambda phage particles as trimers; hybrid DNAs with coslambda of 40 and 47% of the length of lambda phage DNA were packaged as dimers; and those with molecular sizes of 68% of that of lambda phage DNA were packaged mostly as monomers. These results demonstrated that two factors are essential for the packaging of DNAs within lambda phage particles; the presence of coslambda on the DNA molecule and an appropriate size of DNA.

Bacteriocin Plasmids↗