Initial binding of murine leukemia virus particles to cells does not require specific Env-receptor interaction.

The initial step of virus-cell interaction was studied by immunofluorescence microscopy. Single particles of murine leukemia virus (MLV) vectors and human immunodeficiency virus (HIV) were visualized by immunofluorescence. Fluorescent dots representing single virions could be localized by staining of capsid proteins (CA) or surface envelope proteins (SU) after fixation of virus supernatants. This technique can be used to determine particle concentration in viral supernatants and also to study virus-cell interaction. We investigated the role of the Env-receptor interaction for the initial binding event between the cell and the viral particles. Ecotropic MLV vector particles were shown to bind to human cells which do not express the specific viral receptor. In addition, MLV particles defective for Env were shown to bind the cells similarly to infectious MLV. Time course experiments of virus-cell binding and dissociation showed identical profiles for infectious and Env-defective MLV particles and suggested that MLV Env is not involved in the early phases of attachment of virus to cells. The possible implication of cellular factors in enhancing viral binding and infectivity is discussed.

Assembly of Amsacta moorei entomopoxvirus spheroidin into spheroids following synthesis in insect cells using a baculovirus vector.

The gene encoding the major occlusion body protein, spheroidin, of Amsacta moorei entomopoxvirus (AmEPV) was introduced into a baculovirus vector under control of the polyhedrin gene promoter. A recombinant virus produced large, ovoid occlusion body-like structures in both Spodoptera frugiperda and Trichoplusia ni cells. These structures resembled the spheroids found in AmEPV-infected Lymantria dispar cells, except they were devoid of virus particles and were not surrounded by a membrane- or envelope-like structure. These results were confirmed by immunofluoresence microscopy and Western blotting using a specific antipeptide antibody to spheroidin, and suggest that the supramolecular assembly of spheroids is not dependent on other EPV-encoded gene products. Transmission electron microscopy and subcellular fractionation experiments revealed that the spheroid-like structures were assembled in both the nucleus and cytoplasm of the recombinant virus-infected cells. This contrasts with the solely cytoplasmic localization found in AmEPV-infected cells.

Identification of recombinant baculoviruses using green fluorescent protein as a selectable marker.

A rapid procedure for the production and identification of recombinant baculoviruses is described that uses the autofluorescent properties of the Aquorea victoria green fluorescent protein (GFP). Expression of the GFP cDNA (without signal peptide sequence) in Spodoptera frugiperda cells resulted in the synthesis of a 30-kDa protein, which was confirmed as GFP by Western blotting and by the emission of green fluorescence when illuminated with longwave UV light (495 or 365 nm). To use GFP as a marker for the selection of recombinant baculoviruses, we prepared a virus, BacGFP1, in which the GFP cDNA was inserted in lieu of lacZ in BacPAK6. Before the use of BacPAK6 or BacGFP1 in a cotransfection to prepare recombinant baculoviruses, the virus DNA was linearized with Bsu361 to improve the recovery of non-parental virus plaques. The use of BacGFP1 DNA resulted in the recovery of 79%-91% plaques with the non-parental phenotype. Plaques were rapidly identified by simply exposing them briefly to longwave UV light (365 nm) without the need for exogenous substrates or biological stains.

Investigation of the influence of the Mm musculus MyoD1 allele on the efficiency of skeletal muscle regeneration.

MyoD1 alleles distinguish between the Mm musculus and Mm domesticus subspecies of Mus musculus. SJL/J mice, which belong to the Mm musculus subspecies, are able to regenerate skeletal muscle more efficiently than BALB/c mice which possess the Mm domesticus MyoD1 allele. Hence the possibility that regeneration responses are due to species-specific genetic differences including MyoD1 was investigated in this study. Quantitative morphometric analysis following muscle crush injury of 2 Mm musculus strains, MBK and MGL, has indicated that regeneration phenotype is not species-specific and may not be directly related to MyoD1 alleles. These results contrast with prior investigations conducted in SJL/J and BALB/c mice, and indicate that other genes may have a direct influence on skeletal muscle regeneration.

A morphometric technique for the histological quantification of skeletal muscle regeneration.

Variation in the regenerative capacity of damaged skeletal muscle between different strains of mice has been well documented but no precise quantitative method has been established to measure directly the phenotypic influences on muscle regeneration. We have developed such a method which allows clear distinction between the regenerative responses in Quackenbush and BALB/c mice. To quantitate regeneration, crushed tibialis anterior muscles taken from Quackenbush, BALB/c and F2 offspring 10 d postinjury were analysed morphometrically by light microscopy. Myotubes in the intermediate crush zone of transverse muscle sections were abundant in Quackenbush but sparse in BALB/c mice. In F2 offspring, regeneration responses reflected those of the parental strains, with no intermediate phenotypes. Statistical analyses indicated a high level of precision and accuracy in the determination of significant differences between regeneration in the different strains and in the F2 offspring. The data presented indicate that this method of quantification of skeletal muscle regeneration may be used for studies on the assessment of the genetic basis for phenotypic variation.

Polymorphism of the mouse E2A gene due to an intronic deletion of 536 bp.

Examination of genetic polymorphism of the transcription factor-encoding gene E2A in laboratory and wild mice by Southern blotting has revealed the presence of two alleles. The most frequent allele is found in Mus musculus (Mm) musculus, as well as Mm domesticus. The less common allele is restricted to the Mm domesticus subspecies. Characterisation of these alleles has shown that the less common allele contains a deletion of approx. 500 bp located within a 1.8-kb intron immediately upstream from the E12 basic helix-loop-helix exon. DNA sequencing determined the deletion to span 536 bp including nucleotides 1045-1580 of the intron within the common allele. The deleted region includes several sequences with similarity to gene regulatory motifs; however, expression of E12 and intron splicing appeat unaltered. The occurrence of an identical deletion in mice from different geographical regions suggests that the uncommon allele may have a long evolutionary history.

Polymorphism of the murine inhibitor of differentiation-controlling gene, Id.

The structure of Id was examined by Southern analysis in inbred mouse lines which included five subspecies of Mus musculus. No variation was detected within this species. The species Mus cookii shares the same form found in mice of M. musculus derivation, indicative of a long evolutionary history and a common origin. However, five TaqI polymorphisms were found among several Mus species, suggesting that Id has been modified throughout species diversification. Whether these variants impart functional changes is yet to be determined.

Genetic modification of an entomopoxvirus: deletion of the spheroidin gene does not affect virus replication in vitro.

In the late stages of an entomopoxvirus infection, virions become embedded within a crystalline occlusion body or spheroid. Spheroids are composed primarily of a single polypeptide, spheroidin. We describe the construction of a genetically modified Amsacta moorei entomopoxvirus (AmEPV) in which the spheroidin gene coding sequences are deleted and replaced with those of a heterologous reporter gene encoding chloramphenicol acetyltransferase (CAT). A transfer vector, pAmCP1, was prepared containing a unique BamHI site in lieu of the spheroidin gene coding region, together with 1 kbp of upstream and downstream DNA sequence that flanks the spheroidin gene. The flanking sequences provide the transcriptional control signals and also guide homologous recombination so that the spheroidin gene coding region can be replaced with that of the foreign gene. The transfer vector was designed so that the translational start codon of the introduced foreign gene would be utilized. A recombinant virus, AmEPV.CAT, was produced by transfecting AmEPV-infected cells with the transfer vector encoding the CAT gene. The recombinant virus was isolated from wild-type virus by identifying plaques with a spheroidin-negative phenotype. Light microscopy and SDS-PAGE analysis demonstrated that no spheroids or spheroidin protein were produced in the recombinant virus-infected cells. The recombinant virus was able to replicate to high titres (10(7) p.f.u./ml) in insect cells indicating that the spheroidin gene is non-essential for AmEPV replication in vitro. Moderate levels of CAT were synthesized in recombinant virus-infected cells and temporal analyses indicated that CAT synthesis followed the pattern of spheroidin production suggesting that the spheroidin gene promoter was functioning under normal regulatory control in the genetically modified virus.

Evidence for adenine methylation within the mouse myogenic gene Myo-D1.

Previous studies have indicated that there may be uncleavable TaqI sites (TCGA) within the mouse myogenic gene, Myo-D1. Fragments of DNA bearing most of the presumed insensitive TaqI sites have been reproduced using PCR. The presence of each of the originally uncleavable TaqI sites has been confirmed and each TaqI site has been shown to be sensitive to TaqI hydrolysis in PCR-synthesized genomic DNA. Since TaqI is inhibited by methylation of the adenine residue within its recognition sequence (but not by cytosine methylation), it is suggested that specific adenine bases are methylated in the coding region of Myo-DI and maintained throughout cell division. The same TaqI recognition sequences are insensitive to digestion in genomic DNA isolated from various mouse tissues including fetus, regenerating skeletal muscle and a myogenic cell line, all of which express Myo-D1. Thus, adenine methylation is not a modification of DNA following gametic fusion nor does it appear to play a major role in regulation of Myo-D1 expression.

Polymorphism of the mouse transcription factor-encoding gene E2A.

Southern blotting analysis using a cDNA probe consisting of the central portion of the E12 coding region has revealed two distinct forms of E2A, one which is common to all inbred and wild mouse strains derived from Mus musculus musculus and Mus musculus domesticus, whereas the other is less common and has only been found in the wild mouse population of Mus musculus domesticus.

Replication and morphogenesis of Amsacta moorei entomopoxvirus in cultured cells of Estigmene acrea (salt marsh caterpillar).

A study of the sequence of morphogenic events occurring within Amsacta moorei entomopoxvirus-infected Estigmene acrea cells is presented. Stages in virion development, and the various cytopathic effects observed in these cells between 0 and 120 h post-infection (p.i.) are described. Events in the early stages of virion assembly (24 to 48 h p.i.), the formation of the outer viral membrane (48 to 72 h p.i.) and the development of occlusion bodies or spheroids (72 to 120 h p.i.) were identified. Cells grown in TC100 culture medium supported production of mature virus particles, the majority of which were either of the intracellular naked virion form, or mature virions incorporated into occlusion bodies. Only limited production of the extracellular enveloped form was observed in these cells.

Studies on the evolution and function of different forms of the mouse myogenic gene Myo-D1 and upstream flanking region.

The product of the murine Myo-D1 gene is able to initiate the complete sequence of genetic events required for formation of skeletal muscle. Because efficiency of regeneration of skeletal muscle is more pronounced in SJL/J mice, as compared to other strains, differences in the structure of Myo-D1 and the upstream regulatory region were sought to determine whether efficiency of tissue repair was influenced by the structure of the gene itself. Analysis of the restriction-fragment length polymorphism (RFLP) of genomic DNA from SJL/J and different sub-strains of mouse indicated that there are at least three different structural forms of Myo-D1, one of which is unique to SJL/J mice and may have been derived from a double recombinational event involving founder forms of Myo-D1. The unique form of Myo-D1 in SJL/J mice also exhibits a PvuII RFLP upstream from the gene, which may reflect some form of rearrangement or variation in methylation of a potential Myo-D1-binding region. Reference to the size of fragments hybridising with the Myo-D1 probe, following digestion of genomic DNA with TaqI, suggests that in most tissues, adenine residues within Myo-D1 may be extensively methylated. Segregation of Myo-D1 allotypes with response to mechanical injury to skeletal muscle in F2 offspring derived from SJL/J and BALB/c parental strains reveals that increased efficiency of tissue repair is associated with the SJL/J type of Myo-D1 gene. These observations provide new approaches to investigation of genetic control of tissue regeneration and cellular differentiation and proliferation in general.