Predominant and developmentally regulated expression of dynamin in neurons.

We have cloned a cDNA for dynamin, a 100 kd microtubule-associated motor protein whose 5' region contains a GTP-binding motif homologous to that of the Mx proteins, from a rat brain library and analyzed its expression. Dynamin mRNA is 3.6 kb and is preferentially expressed in the brain after postnatal day 7, parallel to the developmental increase of the protein. In situ hybridization revealed high levels of dynamin transcripts in neural cells in the cerebellar cortex, hippocampus (particularly in the CA3 area), and cerebral cortex. The transcripts appeared in cerebellar granular cells only after they had ceased dividing and had migrated to the inner granular layer. We show that dynamin is expressed predominantly in neural cells after elongation of their processes, suggesting a role especially in mature neurons.

Resistance of G mice to murine leukemia virus infection: apparent disparity in in vivo and in vitro resistances.

We observed a marked discordance between in vivo and in vitro sensitivities to Friend murine leukemia virus in G mice. In vivo resistance of G mice was more than 10(5)-fold relative to sensitive DDD mice, whereas in vitro resistance was only 50 to 100-fold. In vivo sensitivity to N- and NB-tropic Friend murine leukemia virus was recessive in (G X DDD)F1 mice, whereas in vitro sensitivity was dominant in the heterozygotes. The resistance of mouse fibroblasts was different from the resistance of fibroblasts of a certain NZB mouse strain.

Surface antigen expressed in hematopoietic cells derived from Fv-4r mouse strains.

Fv-4 locus controls the susceptibility of mice to ecotropic murine leukemia virus (MuLV). Antiserum against Fv-4r/s mice with BALB/c background was raised in inbred BALB/c mice. In the complement-dependent cytotoxicity test, the antiserum killed the hematopoietic cells derived from Fv-4r/- mice but not these from Fv-4s/s mice, and the genetic cross experiments located the locus controlling the expression of the target molecule of the antibody in the close vicinity of Fv-4. In addition, the antigenic expression of the exogenously infecting MuLV suppressed the expression of the target molecule of the antiserum. The expressions of these two antigens appear to compete with each other on the cell surface.

Fv-4: gene controlling resistance to NB-tropic Friend murine leukemia virus. Distribution in wild mice, introduction into genetic background of BALB/c mice, and mapping of chromosomes.

A gene controlling resistance to NB-tropic Friend murine leukemia virus (F-MuLV) was studied in wild mice. Mice of various subspecies were crossed with inbred BALB/c mice, and their F1 hybrids were tested for resistance to F-MuLV. Some mice of Mus musculus molossinus (Japan), M. musculus castaneus (Taiwan and the Philippines), and M. musculus urbanus (Sri Lanka) appeared to have a dominant resistance gene. A partially congenic strain, BALB/c-Fv-4wr (C4W), was established by the introduction of the gene Fv-4wr from a M. musculus molossinus into the genetic background of BALB/c mice. [(C4W X DBA/2) X C57BL/6-Fvs] crosses revealed that Fv-4wr is located on chromosome 12 with the gene order of Fv-4w-Pre-1-lgh-1, apparently at the same site as the Fv-4. The resistance of C4W mice was indistinguishable from the Fv-4-controlled resistance of FRG mice.

Enhancement of 5-iododeoxyuridine-induced endogenous C-type virus activation by polycyclic hydrocarbons: apparent lack of parallelism between enhancement and carcinogenicity.

When mouse MLg cells were treated with 3-methylcholanthrene or 7,12-dimethylbenz[alpha]anthracene in the presence of microsomal enzymes and NADPH after 5-iododeoxyuridine (IUDR) treatment, the induction rate of the endogenous C-type virus was increased fivefold to sixfold in comparison with the culture treated with IUDR only. In this reaction, both the microsomal enzymes and NADPH were indispensable. 7,8-Benzoflavone, an inhibitor of the metabolism of hydrocarbons in hamster embryo cultures, inhibited the reaction. For detecting the enhancing activity, the concentration of IUDR for the pretreatment, the concentration of the test products, and the duration of the treatment with the products were important factors. In screening 30 polycyclic hydrocarbons, we were unable to detect a correlation between the in vivo carcinogenicity in the skin and the enhancing activity in the conditions tested.

Institute profile: National Institute of Infectious Diseases.

A profile of the the National Institute of Infectious Diseases (NIID) at Toyama Shinjuku-Ku, Tokyo, Japan.

A polymorphic repetitive-sequence PR1 family. Evidence for meiotic instability.

When EcoRI digests of mouse genomic DNA were subjected to Southern blot analysis with the polymorphic repetitive sequence PR1 as a probe, one satellite-like band of 3.5 X 10(3) base-pairs, designated as PR1 family B, was detected in BALB/c-strain mice, but not in the DDD/1- or MOA-strain mice. Analysis of recombinant phage clones revealed that the repeating unit of the PR1 family B was 13.5 X 10(3) base-pairs long. This family consisted of a tandem array of repeating units and occupied as much as 2% of one BALB/c chromosome. Since the BALB/c-specific PR1 family B is not present in DDD/1 or MOA mice, the unpaired portion of the BALB/c chromosome may be looped out in a synaptonemal complex during meiosis in F1 hybrids of the BALB/c strain with DDD/1 or MOA. To determine the fate of this extra DNA, we examined the genotypes of the F1 hybrid mice and of the segregating populations. Although the PR1 patterns of F1 and most N2 mice are consistent with typical Mendelian inheritance, some N2 progeny showed an abnormal 3.5 X 10(3) base-pair band of unexpectedly reduced intensity. This indicated that the extra DNA of PR1 family B occasionally underwent recombination during meiosis in F1 mice, resulting in its apparent excision. Examination of PstI digests supported this interpretation.

Novel repetitive sequence families showing size and frequency polymorphism in the genomes of mice.

A middle repetitive sequence, PR1, originally found in mouse rDNA appeared as satellite-like bands when EcoRI and BglII digests of genomic DNA were subjected to Southern blot hybridization using PR1 as probe. The copy number and sizes of PR1-related satellite-like bands, designated as PR1 families, differed remarkably among the subspecies and laboratory strains of mice when the EcoRI digests of genomic DNAs were compared. These bands were not detected in rat and human DNAs. A unit of PR1 sequence was determined by examining cloned EcoRI 3.5 kb (kb, 10(3) bases) fragment and 6.6 kb rDNA by cross-hybridization and sequence analysis: 3.5 kb and 6.6 kb DNAs are composed of homologous PR1 regions and the flanking non-homologous sequences. The results indicate that amplification of different sequences containing PR1 has occurred in different subspecies and strains of mice, and that the segments of satellite-like bands are likely to have been created by recombination of the PR1 sequence with other DNA segments before amplification. The chromosomal distribution of the 3.5 kb PR1 family was studied by back-crossing the female F1 between BALB/c and DDD/1 to male DDD/1. The segregation data strongly suggest that most, if not all, of this family are located on a single chromosome. The stability of these PR1 families in the genomes of cultured cells of a given strain was also examined. An extra band homologous to PR1 appeared in their genomes, but was not detected in other tissues, indicating that some PR1 families may change even during cell propagation.

Homologous recombination involving a large heterology in Escherichia coli.

Recombination between two different deletion alleles of a gene (neo) for neomycin and kanamycin resistance was studied in an Escherichia coli sbcA- recB-C- strain. The two homologous regions were in an inverted orientation on the same plasmid molecule. Kanamycin-resistant plasmids were selected and analyzed. The rate of recombination to form kanamycin-resistant plasmids was decreased by mutations in the recE, recF and recJ genes, but was not decreased by a mutation in the recA gene. It was found that these plasmids often possessed one wild-type kanamycin-resistant allele (neo+) while the other neo allele was still in its original (deletion) form. Among kanamycin-resistant plasmids with one wild-type and one parental allele it was often found that the region between the inverted repeats had been flipped (turned around) with respect to sites outside the inverted repeats. These results were interpreted as follows. Gene conversion, analogous to gene conversion in eukaryotic meiosis, is responsible for a unidirectional transfer of information from one neo deletion allele to the other. The flipping of the region between the inverted repeats is interpreted as analogous to the crossing over associated with gene conversion in eukaryotic meiosis. In contrast with a rec+ strain, these products cannot be explained by two rounds of reciprocal crossing over involving a dimeric form as an intermediate. In the accompanying paper we present evidence that gene conversion by double-strand gap repair takes place in the same E. coli strain.

Orientation dependence in homologous recombination.

Homologous recombination was investigated in Escherichia coli with two plasmids, each carrying the homologous region (two defective neo genes, one with an amino-end deletion and the other with a carboxyl-end deletion) in either direct or inverted orientation. Recombination efficiency was measured in recBC sbcBC and recBC sbcA strains in three ways. First, we measured the frequency of cells carrying neo+ recombinant plasmids in stationary phase. Recombination between direct repeats was much more frequent than between inverted repeats in the recBC sbcBC strain but was equally frequent in the two substrates in the recBC sbcA strain. Second, the fluctuation test was used to exclude bias by a rate difference between the recombinant and parental plasmids and led to the same conclusion. Third, direct selection for recombinants just after transformation with or without substrate double-strand breaks yielded essentially the same results. Double-strand breaks elevated recombination in both the strains and in both substrates. These results are consistent with our previous findings that the major route of recombination in recBC sbcBC strains generates only one recombinant DNA from two DNAs and in recBC sbcA strains generates two recombinant DNAs from two DNAs.

Genetic recombination through double-strand break repair: shift from two-progeny mode to one-progeny mode by heterologous inserts.

Double-strand break repair models of genetic recombination propose that a double-strand break is introduced into an otherwise intact DNA and that the break is then repaired by copying a homologous DNA segment. Evidence for these models has been found among lambdoid phages and during yeast meiosis. In an earlier report, we demonstrated such repair of a preformed double-strand break by the Escherichia coli RecE pathway. Here, our experiments with plasmids demonstrate that such reciprocal or conservative recombination (two parental DNAs resulting in two progeny DNAs) is frequent at a double-strand break even when there exists the alternative route of nonreciprocal or nonconservative recombination (two parental DNAs resulting in only one progeny DNA). The presence of a long heterologous DNA at the double-strand break, however, resulted in a shift from the conservative (two-progeny) mode to the nonconservative (one-progeny) mode. The product is a DNA free from the heterologous insert containing recombinant flanking sequences. The potential ability of the homology-dependent double-strand break repair reaction to detect and eliminate heterologous inserts may have contributed to the evolution of homologous recombination, meiosis and sexual reproduction.

Packageable antiviral therapeutics against human immunodeficiency virus type 1: virion-targeted virus inactivation by incorporation of a single-chain antibody against viral integrase into progeny virions.

To determine their activities as an antiviral agent packageable within virions and suitable for continued expression in cells, we tested a single-chain antibody (scAb) against human immunodeficiency virus type 1 (HIV-1) integrase and its three fusion proteins: fused to viral protein R (scab-Vpr), a double-cassette of the WXXF motif binding to Vpr (scAb-WXXF), and viral major capsid protein (scAb-CA), respectively. Cotransfection of human 293T cells with expression plasmid for scAb-Vpr or -WXXF along with HIV-1 clone pLAI resulted in the production of a normal amount of progeny virions with infectivity decreased by more than 10(3)-fold. Immunoblot analyses showed that scAb-Vpr or -WXXF was associated with virions, whereas scAb or scAb-CA was not, suggesting that scAb-Vpr or -WXXF was incorporated into virions. The incorporation of scAb-WXXF appeared to be Vpr dependent, because the fusion protein was associated with the wild-type but not with Vpr-truncated HIV-1 virions. Since G418-selected HeLa clones carrying expression plasmid for scAb-WXXF were obtained much more frequently than those for scAb-Vpr, scAb-WXXF was inferred to be less toxic to cells than scAb-Vpr. These results suggest that scAb-WXXF may serve as a novel class of antiviral therapeutic that inactivates progeny HIV virions from within.

Involvement of RecE exonuclease and RecT annealing protein in DNA double-strand break repair by homologous recombination.

We demonstrated that a double-stranded (ds) gap in DNA is repaired by a gene conversion mechanism in an Escherichia coli recBC sbcA23 strain, as predicted by the ds break repair models for homologous recombination. The sbcA mutation is known to induce several gene products encoded on the Rac prophage present in most strains of E. coli K-12. These include exonuclease VIII (Exo VIII), a 5' to 3' exonuclease working from the end of a duplex DNA, and RecT, an annealing protein. We found that a rac- strain (lacking the Rac prophage) cannot support this repair. A plasmid carrying part of the Rac prophage supported highly efficient ds gap repair activity in a rac- strain, but two ExoVIII+ recT- plasmids did not. The recE159 mutation that blocks ds gap repair was found to be recT+, since these ExoVIII+ recT- plasmids complemented the recE159 mutation in repair of ultraviolet light damage. From these observations, we conclude that both ExoVIII and RecT are essential for ds gap repair. We discuss their possible roles in the ds break repair reaction.

Characterization and transcriptional regulation of rat glutamine tRNA-encoding genes: repression of tRNA(UUGGln) synthesis.

In rat liver, the amount of tRNA(UUGGln) (RG-2) was found to be approx. 20% of that of tRNA(CUGGln) (RG-1). The independent RG-1 and RG-2 genes were isolated from a rat genomic library together with four RG-related genes containing two to five alterations in their coding regions. Sequence analysis demonstrated that there was no difference between the internal promoter sequences of RG-1 and RG-2. However, interestingly, the transcriptional activity of RG-1 was approximately four-times higher than that of RG-2 in an in vitro transcription reaction. Replacement of the 5'-flanking sequence of RG-2 by the corresponding sequence of RG-1 or by a plasmid DNA sequence caused activation of RG-2 transcription. Gel retardation assay demonstrated that the 5'-flanking region of RG-2 contained a unique sequence specifically recognized by a nuclear protein. Taken together, these results strongly suggest that the transcriptional activity of RG-2 might be negatively regulated by the binding of a nuclear protein at a specific site in the 5'-flanking region of the gene.

Carcinogenic chemicals enhance mouse leukemia virus infection in contact-inhibited culture: a new simple method of screening carcinogens.

Carcinogenic chemicals enhanced infection in contact-inhibited cells with mouse leukemia virus. A correlation was found between the enhancing effects and the carcinogenicities of the 40 chemicals tested. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate did not enhance viral infection.

Alteration of polypeptide synthesis and composition during differentiation of human leukemia cell line HL60.

Using two-dimensional gel electrophoresis, we have analyzed the polypeptide alteration of the human myeloid leukemia cell line HL60 during differentiation induced by dimethylsulfoxide (DMSO) and 12-0-tetradecanoylphorbol-13-acetate (TPA). Three polypeptides increased in synthesis after DMSO treatment, while 5 polypeptides increased after TPA treatment. Most other polypeptides were synthesized at a reduced rate though their net amounts remained unchanged, indicating that myeloid differentiation was associated not only with an increased synthesis of a few specific polypeptides but also with overall depression of most other polypeptides. One of the early differentiation marker proteins (mol. wt. approx. 20,000, pI approx. 6.0) of granulopoietic cells was increased specifically after DMSO treatment, while one of the monocyte specific marker proteins (mol. wt approx. 46,000, pI approx. 4.8) was detected after TPA treatment. But, the pattern of HL60 cells did not necessarily become similar to that of normal mature granulocytes after DMSO treatment, indicating that HL60 cell differentiation might be partial or incomplete.

Virulence phenotypes of Shigella flexneri 2a avirulent mutant 24570 can be complemented by the plasmid-coded positive regulator virF gene.

Avirulent mutation of an opaque colony variant of Shigella flexneri 2a designated 24570 has been believed to be linked with the glpK locus of the chromosome. However, avirulent phenotypes of the 24570 strain could be complemented by the invasion plasmid-coded virF gene, a positive regulator for invasion genes. The 24570 strain had a DNA structural alteration upstream of the virF gene.

Application of the hybridization AT-tailing method for detection of human immunodeficiency virus RNA in cells and simian immunodeficiency virus RNA in formalin-fixed and paraffin-embedded tissues.

An in situ hybridization (ISH) AT-tailing method (HybrAT) was developed for the detection of viral genomes in infected cells and tissues. The method consists of hybridization with oligonucleotide probe which has a 3' end oligo d(A-T) tag, followed by elongation of the oligo d(A-T) by deltaTth DNA polymerase in the presence of the labeled nucleotide. The in situ HybrAT detected human immunodeficiency virus type 1 (HIV-1) in cells and simian immunodeficiency virus (SIV) in formalin-fixed and paraffin embedded sections with a sensitivity comparable to RNA ISH. The advantage of this method over other methods is discussed.

New HIV plaque titration; application to the assay of neutralizing antibody.

A simple, sensitive and accurate plaque assay was developed using HPB-Ma, a variant of the human T-cell line HPB-ALL, which becomes adherent to the substratum after infection with an amphotropic murine sarcoma virus (MSVa). The simplicity of this novel plaque assay allowed us to examine a large number of serum samples from patients with HIV infection for neutralizing antibody activity against two human immunodeficiency virus type-1 (HIV-1) strains. During the progression of clinical disease, the neutralizing activity in the sera from two individual patients remained unchanged or increased. A patient with a known time of HIV infection produced cross-neutralizing antibody at 25-34 weeks. The neutralizing activity in the sera from 17 asymptomatic carriers, four patients with AIDS-related complex and four AIDS patients was also examined and was found to be unrelated to the clinical stage.

A 3D display with a linearly moving mirror to reflect a series of 2D cross sections and its application to noninvasive angiography.

A 3D display equipped with a linearly moving mirror reflecting a series of 2D cross sections pictures has been devised. The pictures were displayed by a CRT in perfect synchronization with the back-and-forth movements of the mirror. The 80x80x80 mm(3) 3D space contained 128x128 pixels; with each pixel having 256 grades of intensity, enough to produce a satisfactory 3D image of an ultrasonic echogram of hepatic vessels. The images can be observed from different angles by changing the observer's position or by prompt rotation of the image through a console. Mechanical problems inherent in using moving parts have been solved.