Neuroattenuation of an avirulent bunyavirus variant maps to the L RNA segment.

The derivation and characterization of a neuroattenuated reassortant clone (RFC 25/B.5) of California serogroup bunyavirus was described previously (M. J. Endres, A. Valsamakis, F. Gonzalez-Scarano, and N. Nathanson, J. Virol. 64:1927-1933, 1990). To map the RNA segment responsible for this attenuation, a panel of reassortants was constructed between the attenuated clone B.5 (genotype TLL) and a virulent clone (B1-1a) of reciprocal genotype (LTT). Parent viruses and clones representing all of the six possible reassortants were examined for neurovirulence by intracerebral injection in adult mice. Reassortants bearing the large RNA segment from the virulent parent were almost as virulent as the virulent parent virus, while reassortants bearing the large RNA segment from the avirulent parent virus exhibited low or intermediate virulence. These results indicate that the large RNA segment is the major determinant of neuroattenuation of clone B.5. In addition to its neuroattenuation, clone B.5 was temperature sensitive and exhibited an altered plaque morphology. These phenotypes also segregated with the large RNA segment. The importance of the large RNA segment (which encodes the viral polymerase) in neurovirulence contrasts with prior studies which indicate that the ability to cause lethal encephalitis after peripheral injection of suckling mice (neuroinvasiveness) is primarily determined by the middle-sized RNA segment, which encodes the viral glycoproteins.     

Isolation and characterization of variant clones of Chinese hamster cells after treatment with irradiated 5-iodouridine.

Variant clones were isolated from cultured Chinese hamster Don cells after treatment with irradiated 5-iodouridine. The following characters of a primary variant clone, C-11 and a secondary variant clone, C-24 were compared with those of the original clone C-1: colony-forming activity, growth rate in the presence of irradiated and unirradiated 5-iodouridine, distribution of chromosome numbers and cell cohesion. The variant clones C-11 and C-24 were partially resistant to unirradiated 5-iodouridine at lower concentration and C-24 cells were slightly resistant to short-term treatment with irradiated 5-iodouridine. Unlike clines C-1 and C-11 the variant clone C-24 showed no lag phase on growth in 5-iodouridine medium. The modal numbers of the chromosomes of all three clones were 22, like that of normal Chinese hamster diploid cells. Of the three clones, the variant C-24 cells showed the least mutual cohesion and the original C-1 cells showed the most. The possibility that an alteration in cellular membrane might be related to an increase in the resistance to radiosensitizing agents are discussed.     

An avirulent G1 glycoprotein variant of La Crosse bunyavirus with defective fusion function.

La Crosse virus, a member of the California serogroup of the family Bunyaviridae, causes encephalitis in humans and laboratory rodents. A variant virus (V22) selected with a monoclonal antibody against the large (G1) glycoprotein showed diminished neuroinvasiveness after peripheral inoculation. This variant has an alteration in its fusion function, requiring a lower pH for the activation of fusion and demonstrating reduced efficiency of cell-to-cell fusion of BHK-21 cultures. V22 was studied in detail following the infection by intraperitoneal or intracerebral routes in suckling, weanling, or adult CD-1 mice. It exhibited a marked reduction in its ability to replicate in striated muscle and to produce viremia; however, after intracerebral injection V22 virus replicated almost as rapidly in brain as its parent, La Crosse virus. V22 virus thus represents an example of reduced neuroinvasiveness associated with an alteration at a specific epitope of the G1 glycoprotein. This same epitope also influences the fusion activity of the glycoprotein.     

Detection and characterization of chimeric yeast artificial-chromosome clones.

Methods for the construction of yeast artificial-chromosome (YAC) clones have been designed to isolate single, large (100-1000 kb) segments of chromosomal DNA. It is apparent from early experience with this cloning system that the major artifact in YAC clones involves the formation of YACs that contain two or more unrelated pieces of DNA. Such "chimeric" YACs are not easily recognized, particularly in libraries constructed from the total DNA of an organism. In some libraries, they have been found to constitute a major fraction of the clones. Here we discuss some of our experiences with chimeric YACs, with particular emphasis on the approaches that we have employed to detect such aberrant clones. In addition, we describe the detailed characterization of one chimeric YAC isolated from a library prepared from total human DNA. The organization of this clone indicates that it formed by in vivo recombination, presumably in yeast, between two Alu sequences located on unrelated segments of human DNA.     

Isolation and properties of DMSO resistant variant clones of Friend leukemia cells.

DMSO resistant clones have been isolated from the inducible Friend leukemia cell line 5-86 both from unmutagenized cultures and following EMS mutagenesis. All the clones can grow in the presence of 1.8% DMSO and are non-inducible or poorly inducible for hemoglobin synthesis by DMSO as well as by other known inducers of Friend leukemia (FL) cells differentiation like hemin, hypoxanthine, hexamethylene bisacetamide. The clones are also defective for the expression of other properties of differentiating Friend cells like agglutinability by plant lectins and expression of the surface protein glycophorin. Some of the clones show an impaired ability to form tumors in vivo. These resistant clones might be useful for a genetic analysis of the differentiation process of Friend leukemia cells.     

Isolation, mapping, and characterization of two cDNA clones expressed in the cerebellum.

In an effort to characterize genes expressed in the cerebellum, we have isolated two cDNA clones, H11B (D16S286) and 507 (D5S344), that hybridized to a cerebellar cDNA probe. Using a panel of human-rodent somatic cell hybrids, cDNA clone H11B was mapped to human chromosome 16, and clone 507 was mapped to human chromosome 5. TaqI RFLPs were identified with both clones and were used for linkage analysis in the CEPH families. D16S286 was tightly linked to several markers near chromosome 16p13, and D5S344 was tightly linked to several markers on chromosome 5q. Sequence tagged sites or expressed sequence tags were generated from the 3' untranslated regions of both cDNA clones.     

Reversibility of malignant transformation as affected by the oncogenic virus SV40. II. The characteristics of virus-induced revertant clones

Fifteen revertant clones exhibiting contact inhibition, one of the typical characteristics of normal cells, were studied after treatment of spontaneously transformed Chinese hamster fibroblasts with SV40. The clones proved to be partial revertants, as regards to other properties of the normal phenotype--loss of the ability to grow in a medium with a low serum content and anchorage-dependence. Viral DNA was detected in all revertant clones. The expression of T-antigen--the product of viral oncogene, was observed in 13 of 15 revertants analyzed. The study of SV40 "rescued" from several revertants in permissive monkey cells has shown that the virus is non-defective. In 7 clones, reversion was accompanied with polyploidization. In the cases, reversion could be due to changes in the balance between oncogenes and suppressor genes (anti-oncogenes). The possibility of induction by SV40 of mutations in anti-oncogenes suppressing the expression of both cellular and viral oncogenes is discussed. It is suggested that reversion to the normal phenotype in clones with a near-diploid karyotype could result from such virus-induced suppressor mutations.     

Human apolipoprotein B: identification of cDNA clones and characterization of mRNA.

Apolipoprotein B (apoB) is a major protein component of low density and very low density lipoproteins. Because of its large size and heterogeneity, molecular studies of apoB have been difficult, and its structure and regulation remain poorly understood. We now report the identification of human apoB cDNA clones by antibody screening of hepatoma libraries in the expression vector lambda gt11. Both oligo(dT) primed and random primed libraries were constructed and screened with polyclonal antibodies to intact apoB, as well as with antibodies raised against a synthetic peptide based on the limited amino acid sequence available for apoB. The identity of the clones was unambiguously established by comparisons of the cloned cDNA sequences with apoB amino acid sequences. The clones hybridize to an exceptionally large 20 kb mRNA that is present in liver and intestine but not other tissues examined, consistent with the distribution expected from protein biosynthetic studies. The properties of the mRNA have implications for the biogenesis of the multiple apoB molecular weight forms secreted by liver and intestine.     

Entamoeba histolytica: generation and characterization of hybrid clones

Transference of DNA to Entamoeba histolytica was carried out by polyethylene glycol fusion of two amebic clones with different phenotypes. Clone C9, strain HM1:IMSS, was the donor. It is emetine-resistant, highly phagocytic and virulent, and grows in soft agar. Clone L6, strain HM1:IMSS, the recipient, is emetine-sensitive, phagocytic, and virulence-deficient, and it does not grow in soft agar. Clones L6 and C9 have shown high stability in their virulence phenotypes since their isolation more than 5 years ago. Before fusion experiments, clone C9 was incubated in 20 micrograms/ml bromodeoxyuridine for 24 hr, and then, irradiated with 310 nm light to complete inactivation. Controls ensured that all irradiated trophozoites died after 24 hr of incubation at 37 degrees C. Irradiated C9 trophozoites were fused with L6 trophozoites, and hybrids were selected by their ability to grow in the presence of emetine. All hybrids, independently generated, grew poorly in soft agar and showed both an intermediate emetine-resistance and rate of phagocytosis. Some of them destroyed efficiently cell monolayers, but interestingly, they showed differences in their ability to produce hepatic abscesses in hamsters.     

Isolation and characterization of cDNA clones for plant cyclins.

We have isolated and sequenced a carrot cDNA and two soybean cDNAs encoding mitotic cyclin homologs. The soybean clones were derived from nearly identical cognate genes. The carrot cyclin and soybean cyclins were slightly more similar to A-type and B-type cyclins thus far defined, respectively. However, they had divergent amino acid sequences in the portion that is most highly conserved in known cyclins and we could not easily include them in either of the phylogenetic types. Since the homology between carrot and soybean cyclins was low, each of them might define a novel and distinct type. The mRNA of carrot cyclin, 1.5 kb in length, was expressed concomitant with somatic embryogenesis of cultured cells. Expression of soybean cyclin mRNAs, 1.6 kb in length, was localized in proliferating parts of seedlings. As in the case of cyclin genes of marine invertebrates, microinjection of a synthetic mRNA for the soybean cyclin induced the maturation of Xenopus oocytes. Other cyclin genes may be present because, on Southern blot analysis of soybean genomic DNA, the isolated soybean cDNA probe hybridized with additional genes under low stringency.     

Rabbit C-reactive protein. Biosynthesis and characterization of cDNA clones

To study the biosynthesis of rabbit C-reactive protein (CRP), a cDNA library was constructed from CRP mRNA-enriched polysomal poly(A) RNA. Four recombinant plasmids, designated pCX9, pCX23, pCX28, and pCX39, from 39 positive clones were sequenced and found to represent overlapping clones. DNA sequencing of CRP cDNA and primer extension of the 5'-end of CRP mRNA have demonstrated that the complete length of rabbit CRP mRNA consists of 2331 nucleotides and a terminal poly(A) segment. Analysis of the resulting sequence indicated that rabbit CRP mRNA contained a 5'-noncoding region of 107 nucleotides, a leader sequence encoding 20 amino acids, a coding region covering 205 amino acids, and a 3'-noncoding region of 1549 nucleotides. The 3'-noncoding region contained a consensus AAUAAA sequence that is 105 nucleotides upstream from the 3'-terminal poly(A) segment. Using an in vitro translation system, we have confirmed that CRP is synthesized as a precursor polypeptide (Mr approximately equal to 26,000) which undergoes processing to form the mature polypeptide (Mr approximately equal to 23,500). The CRP precursor failed to display a calcium-dependent affinity for phosphorylcholine ligand as demonstrated by mature CRP, suggesting that the phosphorylcholine-binding site of CRP only formed after processing. Northern blot analysis suggested that following induction with turpentine, liver was the only site where CRP mRNA synthesis could be demonstrated and that the change in mRNA concentration correlated with the course of CRP production. Southern blot analysis of liver genomic DNA indicated a single gene copy for CRP.     

Trypanosoma gambiense and T. equiperdum: characterization of variant specific antigens

It has been shown that by a simple procedure the variant specific protective antigen can be isolated from both T. gambiense and T. equiperdum. The protection afforded mice by this antigen is relatively long term and a high percentage of mice has complete protection. It has been suggested that the antigens are highly antigenic and although antigenically unique for each relapse strain, they have one or more common physical characteristic. It is therefore hypothesized that a multivalent vaccine might be prepared by similar procedures for protection against trypanosomiasis.     

家养动物体表蜱中发热伴血小板减少综合征布尼亚病毒分离及鉴定

为了解发热伴血小板减少综合征布尼亚病毒(SFTSV)的传播机制,采集了山东疫区家养牛、羊和狗等动物体表蜱,分类鉴定后,通过Real-time PCR筛查、病毒分离培养和基因组序列分析等方法分离鉴定蜱中的病毒。所采集的蜱,以长角血蜱为主,占91.4%。其中3头SFTSV核酸检测阳性,阳性率为2.14%,并在其中一份羊体表蜱标本中分离到SFTSV病毒,命名为SDLZTick12。序列分析显示与我国在不同省份患者标本中分离的病毒全基因序列具有高度同源性,且病毒的抗原性和生长特性与人源病毒相同。本研究首次在山东疫区蜱中分离到新型布尼亚病毒,并与人源病毒进行了系统比较研究,提示蜱可能为该新病原体的传播媒介,对疾病的防控具有重要的指导意义。     

Genetic characterization and relationships of Populus alba,P. tremula,and P. x canescens,and their clones

Summary Isozyme analysis was conducted on individuals of Populus alba L., P. tremula L., and P. × canescens Smith to genetically characterize and differentiate species, hybrids, and individuals, and to determine genetic relationships among them. Thirty gene loci, with 71 alleles, coding for 15 enzymes were observed. Individuals could be identified on the basis of their multilocus genotypes. There were 21 unique multilocus genotypes among 23 P. alba clones. Five P. alba clones from Canada were genetically distinct from each other. Each of the 18 P. tremula and 15 P. × canescens clones had unique multilocus genotypes. Thirteen clones had a unique genotype at a single locus. Percentage of polymorphic loci, average number of alleles per locus, and mean observed heterozygosity were, respectively, 50.0, 1.86, and 0.085 in P. alba, 51.7, 1.66, and 0.096 in P. tremula, and 51.7, 1.86, and 0.157 in P. × canescens. Populus alba and P. tremula were genetically distinct from each other and could be distinguished by mutually exclusive alleles at Aco-3, P. tremula-specific gene Mdh-3, and allele frequency differences at 6 loci. Populus × canescens had allele contributions of P. alba and P. tremula. However, their allele frequencies were closer to those of P. alba than being truly intermediate. The mean genetic identity was 0.749 between P. alba and P. tremula, 0.987 between P. alba and P. × canescens, and 0.817 between P. tremula and P. × canescens. Canonical discriminant analysis of multilocus genotypes separated P. alba, P. tremula, and P. × canescens into three distinct groups and portrayed similar interspecific relationship as above. Our results suggested that the putative P. × canescens individuals consisted of a mixture of F₁ hybrids of P. alba and P. tremula and their backcrosses to P. alba.Presently with the University of Alberta, and BioGenetica Inc., P.O. Box 8261, Edmonton, Alberta, Canada T6H 4P1     

S49 lymphoma wild type and variant clones contain normal calcium dependent regulator.

Calcium dependent regulator is present in wild-type S49 lymphoma cells, in the variant that is deficient in adenylate cyclase activity (AC-), and in the uncoupled variant (UNC). The electrophoretic mobility and the ability to stimulate cyclic nucleotide phosphodiesterase of the calcium dependent regulator from each of these three clones are indistinguishable from those of the modulator protein isolated from bovine brain. Calcium dependent regulator does not appear to be involved in the defect responsible for the UNC or AC- variants.     

Methionine metabolism in BHK cells: selection and characterization of ethionine resistant clones.

The selection of clones resistant to methionine antagonists was undertaken on baby hamster Kidney cells grown in a methionine free medium, supplemented with homocystine, folic acid and hydroxo-B12. Clones resistant to 30 mug/ml ethionine were isolated after mutagenesis at an induced mutation frequency of 2.3 X 10(-5). An ethionine resistant clone, ETH 304, was extensively studied. The resistant cells excreted methionine in the culture medium and the intracellular pools of methionine and SAM were two to five times greater in the resistant clone than in the wild type cells. A semidominant ethionine resistant phenotype was observed in hybrids between the wild type and this resistant clone. Measurement of the specific activity of menadione reductase, B12 methyltransferase and ATP: L-methionine S-adenosyl-transferase in crude extracts of the wild type showed a repressive action of methionine on the level of the three enzymes. However, the ethionine resistant clone ETH 304 was not modified in this function. Menadione reductase is feedback-inhibited by SAM in wild type cells. The enzyme of the ethionine resistant clone was significantly less sensitive to SAM. When a comparison of thermal stability was made between the wild type and ethionine resistant clone enzymes, it was found that the thermal stability of the latter was modified. Three other ethionine resistant clones, independantly isolated, were similarly affected in the properties of menadione reductase. These results suggest that the pathway of re-use of S-adenosyl homocysteine, produced during methylation reactions, is highly regulated by methionine and SAM.     

Identification and characterization of cDNA clones encoding hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase from tobacco.

The sequences of three cDNA clones that include the complete coding region of hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase (THT) from tobacco are reported. The three cDNAs were isolated by antibody screening of a cDNA expression library produced from poly(A)+RNA purified from tobacco leaves (Nicotiana tabacum cv. Bottom Special), previously infiltrated with an incompatible strain of Ralstonia solanacearum. The identity of these clones was confirmed by the detection of THT activity in extracts of transformed Escherichia coli and by matching the translated polypeptides with tryptic enzyme sequences. cDNA clones tht4 and tht11 differ only by their 5' leader and 3' UTRs and therefore encode the same protein, whereas tht10 and tht11 exhibit 95 and 99% sequence identity at the DNA and deduced amino acid levels, respectively. The three clones encode proteins of 226 amino acids with calculated molecular masses of 26 kDa. The deduced amino acid sequences show no similarity with the sequence of anthranilate hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus, the only enzyme exhibiting hydroxycinnamoyltransferase activity to be cloned so far in plants. In contrast, comparison of the THT amino acid sequence with protein sequence databases revealed substantial homology with mammalian diamine acetyltransferases. The THT clones hybridized to a 0.95-kb mRNA from elicited tobacco cell-suspension cultures and also to a mRNA of similar size from wound-healing potato tubers. The messengers for THT were also found to be expressed at relatively high levels in tobacco root tissues. Southern hybridization of tobacco genomic DNA with THT cDNA suggests that several copies of the THT gene occur in the tobacco genome. Inhibition experiments using amino-acid-specific reagents demonstrated that both histidyl and cysteyl residues are required for THT activity. In the course of these experiments THT was also found to be inhibited by (2-hydroxyphenyl) amino sulfinyl acetic acid 1,1-dimethylethyl ester, an irreversible inhibitor of cinnamyl alcohol dehydrogenase.