Cells producing Rous sarcoma virus contain virus-specific ribonucleic acid (RNA) which can be identified by hybridization to single-stranded deoxyribonucleic acid (DNA) synthesized with RNA-directed DNA polymerase. Hybridization was detected by either fractionation on hydroxyapatite or hydrolysis with single strand-specific nucleases. Similar results were obtained with both procedures. The hybrids formed between enzymatically synthesized DNA and viral RNA have a high order of thermal stability, with only minor evidence of mismatched nucleotide sequences. Virus-specific RNA is present in both nuclei and cytoplasm of infected cells. This RNA is remarkably heterogeneous in size, including molecules which are probably restricted to the nucleus and which sediment in their native state more rapidly than the viral genome. The nature of the RNA found in cytoplasmic fractions varies from preparation to preparation, but heterogeneous RNA (ca. 4-50S), smaller than the viral genome, is always present in substantial amounts. MATERIALS AND METHODS Materials. The sources of most reagents have been described (13, 15). Conidia of N. crassa were purchased from Miles Laboratories, Inc.; dimethylsulfoxide from Matheson, Coleman and Bell; Takadiastase (Sanzyme) was a gift from Sankyo Ltd., Tokyo, Japan. Diastase powder obtained from Sigma also contains S-1 nuclease, but the data in the present communication pertain only to the Sankyo material. All cell cultures were prepared from embryos known to be free from carrier infection with avian leukosis virus (embryonated eggs obtained from Kimber Farms, Berkeley, Calif.). Electrophoretically purified deoxyribonuclease was purchased from Worthington Biochemicals and treated with iodoacetate by the method of Zimmerman and Sandeen (34) to inactivate traces of contaminating ribonuclease. The alkylated preparations were tested for ribonuclease with 32p_ labeled 70S RSV RNA, which was denatured (11, 12) after exposure to the enzyme and was analyzed by rate-zonal centrifugation. 891

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The protamines are small, basic, arginine-rich proteins synthesized postmeiotically in the testes. Analysis of the regulation of synthesis of the protamine mRNA and protein is restricted by the difficulty in culturing and manipulating the cells in which transcription and translation occur. To avoid these problems, we have produced transgenic mice carrying fusion genes in which sequences 5' to the mouse protamine-2 gene have been linked to exons 2 and 3 of the mouse c-myc gene and, separately, to the simian virus 40 (SV40) early region. We show here that the prot.myc gene is correctly regulated; transcription is detected only in the round spermatids. In one family of transgenic mice carrying the 5' protamine-SV40 T-antigen fusion gene, SV40 early-region mRNA accumulated to the highest level in the testes but was also detected in the thymuses, brains, hearts, and preputial glands of the animals. Although we have demonstrated specific transcription of these fusion genes in the round spermatids, we were not able to detect the SV40 T-antigen protein.During spermatogenesis in mammals, the histones are ultimately replaced by protamines, which are small, basic proteins containing 40 to 70% arginine (for reviews, see references 6, 17, and 18). This change occurs during the late-haploid phases of male germ cell differentiation and produces a DNA-protamine complex that is compact and genetically inactive. The mammalian protamines are translated from RNA transcripts that are detectable only in the haploid cells of the testes.The nuclei of all mammalian sperm examined contain the P1 protamine, and a comparison of the complete sequence analysis of P1 protamines from several species has revealed identical lengths of 50 amino acids and strong sequence homologies (2, 10, 22-25, 28, 29, 35, 38). However, in mice and hamsters there are at least two distinct protamines, and in humans there are three protamine variants (2,3,24,25,27,30). The mouse P1 protamine, mPl, is similar to the P1 protamines of other mammalian species and is synthesized as the mature protein (22). The mouse P2 protamine, mP2, is distinguished from mPl by its amino acid composition: mPl has no histidine residues, whereas mp2 has 13 histidine residues (41). In addition, mP2 is synthesized as a larger protein of 106 amino acids that is processed into the mature protein of 63 amino acids found in spermatozoa (2, 41).The genes for the mouse protamines are present as singlecopy genes on chromosome 16 (19). They are transcribed only in the testis; the mRNAs are first detected in the round spermatids, the first haploid precursors of mature spermatozoa (20,21). These stable, very abundant mRNAs are then stored for up to 8 days before being translated in the elongating spermatids. A shortening of the poly(A) tail coincides with the presence of the mRNAs on polysomes (21). Mouse spermatids contain the two variant protamines in a ratio of approximately 1:1, but in the maturation process the ratio changes to 2:1 (mP2 to mPl) for the mature sperm (3). Whether these changes ...

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which causes devastating epizootics of trout and salmon fry in hatcheries around the world. In laboratory and field studies, epizootic survivors are negative for infectious virus by plaque assay at about 50 days postexposure. Survivors are considered virus free with no sequelae and, thus, are subsequently released into the wild. When adults return to spawn, infectious virus can again be isolated. Two hypotheses have been proposed to account for the source of virus in these adults. One hypothesis contends that virus in the epizootic survivors is cleared and that the adults are reinfected with IHNV from a secondary source during their migration upstream. The second hypothesis contends that IHNV persists in a subclinical or latent form and the virus is reactivated during the stress of spawning. Numerous studies have been carried out to test these hypotheses and, after 20 years, questions still remain regarding the maintenance of IHNV in salmonid fish populations. In the study reported here, IHNV-specific lesions in the hematopoietic tissues of rainbow trout survivors, reared in specific-pathogen-free water, were detected 1 year after the epizootic. The fish did not produce infectious virus. The presence of viral protein detected by immunohistochemistry, in viral RNA by PCR amplification, and in IHNV-truncated particles by immunogold electron microscopy confirmed the presence of IHNV in the survivors and provided the first evidence for subclinical persistence of virus in the tissues of IHNV survivors.

Two rainbow trout (Oncorhynchus mykiss) Mx cDNAs were cloned by using RACE (rapid amplification of cDNA ends) PCR and were designated RBTMx2 and RBTMx3. The deduced RBTMx2 and RBTMx3 proteins were 636 and 623 amino acids in length with molecular masses of 72 and 70.8 kDa, respectively. These proteins, along with the previously described RBTMx1 protein (G. D. Trobridge and J. A. Leong, J. Interferon Cytokine Res. 15:691-702, 1995), have between 88.7 and 96.6% identity at the amino acid level. All three proteins contain the tripartite GTP binding domain and leucine zipper motif common to Mx proteins. A monospecific polyclonal antiserum to an Escherichia coli-expressed fragment of RBTMx3 was generated, and that reagent was found to react with all three rainbow trout Mx proteins. Subsequently, endogenous Mx production in RTG-2 cells induced with poly(IC) double-stranded RNA was detected by immunoblot analysis. The cellular localization of the rainbow trout proteins was determined by transient expression of the RBTMx cDNAs in CHSE-214 (chinook salmon embryo) cells. A single-cell transient-transfection assay was used to examine the ability of each Mx cDNA clone to inhibit replication of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV). No significant inhibition in the accumulation of the IHNV nucleoprotein was observed in cells expressing either trout Mx1, Mx2, or Mx3 in transiently transfected cells.