Genome-wide linkage disequilibrium and past effective population size in three Korean cattle breeds.

The routine collection and use of genomic data are useful for effectively managing breeding programs for endangered populations. Linkage disequilibrium (LD) using high-density DNA markers has been widely used to determine population structures and predict the genomic regions that are associated with economic traits in beef cattle. The extent of LD also provides information about historical events, including past effective population size (Ne ), and it allows inferences on the genetic diversity of breeds. The objective of this study was to estimate the LD and Ne in three Korean cattle breeds that are genetically similar but have different coat colors (Brown, Brindle and Jeju Black Hanwoo). Brindle and Jeju Black are endangered breeds with small populations, whereas Brown Hanwoo is the main breeding population in Korea. DNA samples from these cattle breeds were genotyped using the Illumina BovineSNP50 Bead Chip. We examined 13 cattle breeds, including European taurines, African taurines and indicines, and hybrids to compare their LD values. Brown Hanwoo consistently had the lowest mean LD compared to Jeju Black, Brindle and the other 13 cattle breeds (0.13, 0.19, 0.21 and 0.15-0.22 respectively). The high LD values of Brindle and Jeju Black contributed to small Ne values (53 and 60 respectively), which were distinct from that of Brown Hanwoo (531) for 11 generations ago. The differences in LD and Ne for each breed reflect the breeding strategy applied. The Ne for these endangered cattle breeds remain low; thus, effort is needed to bring them back to a sustainable tract.

Models for Estimating Genetic Parameters of Milk Production Traits Using Random Regression Models in Korean Holstein Cattle.

The objectives of the study were to estimate genetic parameters for milk production traits of Holstein cattle using random regression models (RRMs), and to compare the goodness of fit of various RRMs with homogeneous and heterogeneous residual variances. A total of 126,980 test-day milk production records of the first parity Holstein cows between 2007 and 2014 from the Dairy Cattle Improvement Center of National Agricultural Cooperative Federation in South Korea were used. These records included milk yield (MILK), fat yield (FAT), protein yield (PROT), and solids-not-fat yield (SNF). The statistical models included random effects of genetic and permanent environments using Legendre polynomials (LP) of the third to fifth order (L3-L5), fixed effects of herd-test day, year-season at calving, and a fixed regression for the test-day record (third to fifth order). The residual variances in the models were either homogeneous (HOM) or heterogeneous (15 classes, HET15; 60 classes, HET60). A total of nine models (3 orders of polynomials×3 types of residual variance) including L3-HOM, L3-HET15, L3-HET60, L4-HOM, L4-HET15, L4-HET60, L5-HOM, L5-HET15, and L5-HET60 were compared using Akaike information criteria (AIC) and/or Schwarz Bayesian information criteria (BIC) statistics to identify the model(s) of best fit for their respective traits. The lowest BIC value was observed for the models L5-HET15 (MILK; PROT; SNF) and L4-HET15 (FAT), which fit the best. In general, the BIC values of HET15 models for a particular polynomial order was lower than that of the HET60 model in most cases. This implies that the orders of LP and types of residual variances affect the goodness of models. Also, the heterogeneity of residual variances should be considered for the test-day analysis. The heritability estimates of from the best fitted models ranged from 0.08 to 0.15 for MILK, 0.06 to 0.14 for FAT, 0.08 to 0.12 for PROT, and 0.07 to 0.13 for SNF according to days in milk of first lactation. Genetic variances for studied traits tended to decrease during the earlier stages of lactation, which were followed by increases in the middle and decreases further at the end of lactation. With regards to the fitness of the models and the differential genetic parameters across the lactation stages, we could estimate genetic parameters more accurately from RRMs than from lactation models. Therefore, we suggest using RRMs in place of lactation models to make national dairy cattle genetic evaluations for milk production traits in Korea.

Genetic parameters for yearling weight, carcass traits, and primal-cut yields of Hanwoo cattle.

Genetic parameters associated with yearling weight, carcass traits, and primal-cut yields of male Hanwoo cattle were investigated using univariate and bivariate animal models. The mean yearling weight (YWT), carcass weight (CWT), longissimus muscle area (LMA), backfat thickness (BFT), and marbling score (MS) were 352.47 ± 0.40 kg, 337.39 ± 0.64 kg, 78.28 ± 0.13 cm2, 8.45 ± 0.05 mm, and 3.25 ± 0.03, respectively. Total primal-cut yield (TPC) was 78.95 ± 0.10% of CWT, of which 42.3% was contributed by the forequarters (chuck, CHK; shoulder, SLD; ribs, RIB; and brisket and flank, BAF). Loins, top round (TRND), and round (RND) were associated with yields of 13.57%, 5.45 ± 0.01%, and 8.87 ± 0.02%, respectively. The largest cut studied was ribs (15.67 ± 0.03%). The estimated heritabilities (h2) of YWT, CWT, LMA, BFT, and MS were 0.18 ± 0.02, 0.29 ± 0.04, 0.38 ± 0.05, 0.45 ± 0.05, and 0.62 ± 0.07, respectively. Shoulder yield was highly heritable in Hanwoo steers (0.83 ± 0.13), followed by the yields of round (0.66 ± 0.12), striploin (0.64 ± 0.12), top round (0.62 ± 0.12), sirloin (0.60 ± 0.12), and total primal-cut yield (0.52 ± 0.11). The h2 values of CHK, BAF, RIB, and tenderloin (TLN) ranged from 0.19 ± 0.09 to 0.41 ± 0.11. Generally, the genetic CV was low for most traits (2.33%-6.15%), except for CHK, BFT, and MS. The genetic correlation (rg) was strong between YWT and CWT (0.77 ± 0.06). The greatest positive and negative rg among carcass traits were those between LMA and CWT (0.52 ± 0.08) and between LMA and BFT (-0.30 ± 0.09), respectively. The correlation between CHK and SLD (0.81 ± 0.14), and those between SLD, TLN, TRND, and RND, were mostly strong (0.77-0.87), but the rg between RIB and other traits were strongly negative. The TPC yield showed moderate to high rg with most primal cuts. The YWT, CWT, and LMA correlated notably with CHK, SLD, and loin yields, especially LMA. However, BFT and MS were negatively correlated with many primal cuts but RIB. Those rg estimates were also opposite of that of LMA and CWT with primal cuts. Phenotypic correlations (rp) were generally weaker than rg estimates. The rp of YWT, CWT, and LMA were either zero or moderately negative compared to those of the BFT and MS with primal cuts. Most primal cuts yielded positive rp estimates among them, except for RIB. Our results suggest that direct selection for YWT, various carcass traits, and primal-cut yields may increase the carcass value of Hanwoo males.

Estimation of Genetic Parameters for Somatic Cell Scores of Holsteins Using Multi-trait Lactation Models in Korea.

The study was conducted to analyze the genetic parameters of somatic cell score (SCS) of Holstein cows, which is an important indicator to udder health. Test-day records of somatic cell counts (SCC) of 305-day lactation design from first to fifth lactations were collected on Holsteins in Korea during 2000 to 2012. Records of animals within 18 to 42 months, 30 to 54 months, 42 to 66 months, 54 to 78 months, and 66 to 90 months of age at the first, second, third, fourth and fifth parities were analyzed, respectively. Somatic cell scores were calculated, and adjusted for lactation production stages by Wilmink's function. Lactation averages of SCS (LSCS1 through LSCS5) were derived by further adjustments of each test-day SCS for five age groups in particular lactations. Two datasets were prepared through restrictions on number of sires/herd and dams/herd, progenies/sire, and number of parities/cow to reduce data size and attain better relationships among animals. All LSCS traits were treated as individual trait and, analyzed through multiple-trait sire models and single trait animal models via VCE 6.0 software package. Herd-year was fitted as a random effect. Age at calving was regressed as a fixed covariate. The mean LSCS of five lactations were between 3.507 and 4.322 that corresponded to a SCC range between 71,000 and 125,000 cells/mL; with coefficient of variation from 28.2% to 29.9%. Heritability estimates from sire models were within the range of 0.10 to 0.16 for all LSCS. Heritability was the highest at lactation 2 from both datasets (0.14/0.16) and lowest at lactation 5 (0.11/0.10) using sire model. Heritabilities from single trait animal model analyses were slightly higher than sire models. Genetic correlations between LSCS traits were strong (0.62 to 0.99). Very strong associations (0.96 to 0.99) were present between successive records of later lactations. Phenotypic correlations were relatively weaker (<0.55). All correlations became weaker at distant lactations. The estimated breeding values (EBVs) of LSCS traits were somewhat similar over the years for a particular lactation, but increased with lactation number increment. The lowest EBV in first lactation indicated that selection for SCS (mastitis resistance) might be better with later lactation records. It is expected that results obtained from these multi-trait lactation model analyses, being the first large scale SCS data analysis in Korea, would create a good starting step for application of advanced statistical tools for future genomic studies focusing on selection for mastitis resistance in Holsteins of Korea.

Development of microsatellite markers for the kelp grouper Epinephelus bruneus by 454 pyrosequencing and transfer to related species.

The kelp or longtooth grouper (Epinephelus bruneus), which inhabits Eastern Asia, is the most economically important of 11 grouper species that inhabit the Southern Sea near Jeju Island in Korea. This species is listed as vulnerable by the International Union for the Conservation of Nature and Natural Resources because of a rapid decrease in its resources. We developed microsatellite markers for E. bruneus using the pyrosequencing technique for applications in resource management and aquaculture. In addition, we tested the cross-species transferability of the microsatellite markers in four species belonging to the Epinephelus genus. Among 66,452 simple sequence repeats, 64 loci containing more than eight CA or TG repeats were randomly selected for primer synthesis; 45 primer sets (75.0%) produced polymerase chain reaction (PCR) products of 100-300 bp and were selected as candidates. After primary testing with four E. bruneus fish, 28 polymorphic loci were selected as the final microsatellite markers, and 23 sets showing clear amplification of polymorphic loci were used to analyze 71 fish. These loci have allele numbers ranging from 2 to 23. Null alleles were detected at three loci, and three loci showed an excess of homozygotes in the Hardy-Weinberg equilibrium test. Of the three species used for cross-species transfer of these markers, Epinephelus moara showed the highest transferability (92.9%) and polymorphism (67.9%), followed by Epinephelus fuscoguttatus (75.0 and 67.9%, respectively) and Epinephelus septemfasciatus (57.1 and 46.4%, respectively). These results suggested that these microsatellite loci should be valuable tools for population genetic studies of the species Epinephelus.

Development of microsatellite markers for a hard-shelled mussel, Mytilus coruscus, and cross-species transfer.

The Korean mussel Mytilus coruscus, an endemic marine bivalve mollusk, is economically important. Its population is currently decreasing due to overexploitation and invasion of a more competitive species, Mytilus galloprovincialis. In this study, microsatellite markers for M. coruscus were developed using a cost-effective pyrosequencing technique. Among the 33,859 dinucleotide microsatellite sequences identified, 176 loci that contained more than 8 CA, CT, or AT repeats were selected for primer synthesis. Sixty-four (36.4%) primer sets were produced from the 100- to 200-bp polymerase chain reaction products obtained from 2 M. coruscus individuals. Twenty of these were chosen to amplify DNA from 82 M. coruscus individuals, and 18 polymorphic loci and 2 monomorphic loci were selected as microsatellite markers. The number of alleles and the allele richness of the polymorphic loci ranged from 2 to 22 and from 2.0 to 19.7 with means of 10.8 and 10.1, respectively. Null alleles were detected for all but three loci, which resulted in an observed heterozygosity lower than the expected heterozygosity and therefore an excess of homozygotes. In a cross-species transfer analysis of these markers using 7 Mytilidae species, the locus Mc65 was amplified from all species tested and was found to be polymorphic in all of them. Among the species, M. galloprovincialis, Lithophaga curta, and Hormomya mutabilis showed the same transferability of 25%, but the five amplified loci were polymorphic only in M. galloprovincialis and H. mutabilis. These microsatellite markers may be useful for future resource management and artificial production of juveniles for aquaculture.

Effect of carcass traits on carcass prices of holstein steers in Korea.

The present study investigated the contribution of carcass traits on carcass prices of Holstein steers in Korea. Phenotypic data consisted of 76,814 slaughtered Holsteins (1 to 6 yrs) from all over Korea. The means for live body weight at slaughter (BWT), chilled carcass weight (CWT), dressing percentage (DP), quantity grade index (QGI), eye muscle area (EMA), backfat thickness (BF) and marbling score (MS), carcass unit price (CUP), and carcass sell prices (CSP) were 729.0 kg, 414.2 kg, 56.79%, 64.42, 75.26 cm(2), 5.77 mm, 1.98, 8,952.80 Korean won/kg and 3,722.80 Thousand Korean won/head. Least squares means were significantly different by various age groups, season of slaughter, marbling scores and yield grades. Pearson's correlation coefficients of CUP with carcass traits ranged from 0.12 to 0.62. Besides, the relationships of carcass traits with CSP were relatively stronger than those with CUP. The multiple regression models for CUP and CSP with carcass traits accounted 39 to 63% of the total variation, respectively. Marbling score had maximum economic effects (partial coefficients) on both prices. In addition, the highest standardized partial coefficients (relative economic weights) for CUP and CSP were calculated to be on MS and CWT by 0.608 and 0.520, respectively. Path analyses showed that MS (0.376) and CWT (0.336) had maximum total effects on CUP and CSP, respectively; whereas BF contributed negatively. Further sub-group (age and season of slaughter) analyses also confirmed the overall outcomes. However, the relative economic weights and total path contributions also varied among the animal sub-groups. This study suggested the significant influences of carcass traits on carcass prices; especially MS and CWT were found to govern the carcass prices of Holstein steers in Korea.

Genetic differentiation of octopuses from different habitats near the Korean Peninsula and eastern China based on analysis of the mDNA cytochrome C oxidase 1 gene.

Distributed along the coastal waters of Korea and China, Octopus minor is found in various habitats, including the mud flats in the southern and western coasts of the Korean Peninsula and the rocky areas around Jeju Island; however, the genetic relationships among the different populations are unknown and have not been studied. We compared 630-nucleotide sequences of the CO1 gene from O. minor specimens collected from five regions around the Korean Peninsula and three regions from eastern China in order to determine population structure and genetic relationships. Based on the sequences at 12 polymorphic sites in this region, 11 haplotypes were identified from 85 specimens. Individuals from Jeju Island had unique haplotypes, including two haplotypes not found in the other populations. Nucleotide and haplotype diversity for all populations ranged from 0.03-0.37 and 0.20-0.64, respectively. Pairwise F(ST) values indicated significant genetic differences in populations from Korea and China. An UPGMA dendrogram showed separation of the eight populations into three clusters; one included only the Jeju population, another included the rest of the Korean populations and some from Dalian, China; a third cluster consisted of two other populations from China. We conclude that there are discrete genetic differences in O. minor from the different habitats, suggesting that the populations should be considered as management units in the ongoing recovery program.

Determination of Genetic Diversity among Korean Hanwoo Cattle Based on Physical Characteristics.

This study was conducted to establish genetic criteria for phenotypic characteristics of Hanwoo cattle based on allele frequencies and genetic variance analysis using microsatellite markers. Analysis of the genetic diversity among 399 Hanwoo cattle classified according to nose pigmentation and coat color was carried out using 22 microsatellite markers. The results revealed that the INRA035 locus was associated with the highest Fis (0.536). Given that the Fis value for the Hanwoo INRA035 population ranged from 0.533 (white) to 1.000 (white spotted), this finding was consistent with the loci being fixed in Hanwoo cattle. Expected heterozygosities of the Hanwoo groups classified by coat colors and degree of nose pigmentation ranged from 0.689±0.023 (Holstein) to 0.743±0.021 (nose pigmentation level of d). Normal Hanwoo and animals with a mixed white coat showed the closest relationship because the lowest DA value was observed between these groups. However, a pair-wise differentiation test of Fst showed no significant difference among the Hanwoo groups classified by coat color and degree of nose pigmentation (p<0.01). Moreover, results of the neighbor-joining tree based on a DA genetic distance matrix within 399 Hanwoo individuals and principal component analyses confirmed that different groups of cattle with mixed coat color and nose pigmentation formed other specific groups representing Hanwoo genetic and phenotypic characteristics. The results of this study support a relaxation of policies regulating bull selection or animal registration in an effort to minimize financial loss, and could provide basic information that can be used for establishing criteria to classify Hanwoo phenotypes.

Microsatellite analysis as a tool for discriminating an interfamily hybrid between olive flounder and starry flounder.

An interspecific artificial hybrid was produced between two economically important aquaculture flatfish: olive flounder (Paralichthys olivaceus) and starry flounder (P. stellatus). This hybrid displays the rapid growth characteristic of the former and tolerance to low temperatures and low salinity of the latter, but the genetics of inheritance in this hybrid have not been elucidated. Polymorphic microsatellite markers developed for P. olivaceus and P. stellatus were tested to determine if these markers can be used for analysis of parentage and genetic inheritance. Multiplex PCR using two primer sets that were specific to each species produced PCR products of different sizes; these could be used for the identification of interspecific hybrids. Among the 192 primers derived from olive flounder, 25.5% of the primer sets successfully amplified genomic DNA from starry flounder, and 23% of the 56 primer sets originating from starry flounder amplified DNA from olive flounder. Analysis of genetic inheritance in the hybrid using seven of the 62 microsatellite markers common to both species demonstrated classic Mendelian inheritance of these markers in the hybrid progeny, with the exception of one locus identified as a null allele in the hybrid. These results demonstrate that cross-specific microsatellite markers can be used tools for parentage analysis of hybrid flatfish, for mapping quantitative trait loci, for marker-assisted selective breeding, and for studies of the evolution of fish.

Development of genetic markers in abalone through construction of a SNP database.

In the absence of a reference genome, single-nucleotide polymorphisms (SNP) discovery in a group of abalone species was undertaken by random sequence assembly. A web-based interface was constructed, and 11 932 DNA sequences from the genus Haliotis were assembled, with 1321 contigs built. Of these, 118 contigs that consisted of at least ten annotation groups were selected. The 1577 putative SNPs were identified from the 118 contigs, with SNPs in several HSP70 gene contigs confirmed by PCR amplification of an 809-bp DNA fragment. SNPs in the HSP70 gene were compared across eight abalone species. A total of 129 polymorphic sites, including heterozygote sites within and among species, were observed. Phylogenetic analysis of the partial HSP70 gene region showed separation of the tested abalone into two groups, one reflecting the southern hemisphere species and the other the northern hemisphere species. Interestingly, Haliotis iris from New Zealand showed a closer relationship to species distributed in the northern Pacific region. Although HSP genes are known to be highly conserved among taxa, the validation of polymorphic SNPs from HSP70 in this mollusc demonstrates the applicability of cross-species SNP markers in abalone and the first step towards universal nuclear markers in Haliotis.

Molecular variation in pigmentation genes contributing to coat colour in native Korean Hanwoo cattle.

Pigmentation genes such as TYR (tyrosinase), TYRP1 (tyrosinase-related protein 1), DCT (previously TYRP2, or tyrosinase-related protein 2), ASIP (agouti) and MC1R (melanocortin receptor 1) play a major role in cattle coat colour. To understand the genotypic profile underlying coat colour in native Korean Hanwoo cattle and Angus black cattle, portions of the above-mentioned genes were amplified. Sequence analysis revealed variation in the TYRP1 (exon 5) and MC1R genes. Restriction enzyme analysis of these two genes could distinguish between different colours of Hanwoo cattle. Quantitative estimates of melanin and eumelanin in hair from three different-coloured Hanwoo phenotypes and Angus black showed significant differences at the breed and phenotypic levels. Finally, sequence variants in MC1R were associated with total melanin and eumelanin in breeds as well as in Hanwoo phenotypes.

Cloning and sequence analysis of the interleukin-8 gene from flounder (Paralichthys olivaceous).

A cDNA library of mRNA from flounder leukocytes stimulated with bacterial lipopolysaccharide (LPS) and hemagglutinin was constructed to clone cytokine genes of this fish. Initial screening of this library with human cytokine gene probes was not productive and clones with inserts of over 400 nucleotides (nt) were randomly sequenced, and a homologue of the vertebrate interleukin-8 (IL-8) gene was isolated. The flounder IL-8 cDNA encompassed 884 nt, including a coding region of 330 nt. Four cysteines characteristic of CXC chemokines were identified at conserved locations in the putative protein. The deduced amino acid sequence showed 36 and 35% sequence identity with counterpart genes in monkey and human, respectively, and 52% sequence similarity with these genes. However, the putative flounder IL-8 amino acid sequence showed 25% identity and 52% similarity to that of lamprey, the only other piscine IL-8 gene that has been cloned. Flounder IL-8 transcripts were detected in the head-kidney and spleen of LPS-injected flounder and leukocytes stimulated with LPS. It was not detected in the muscle or liver of LPS-injected flounder, tissues taken from non-stimulated flounder and non-stimulated leukocytes.

CDNA for an immune response gene encoding low molecular weight polypeptide from flounder, Paralichthys olivaceus.

The cDNA for an immune response gene encoding the low molecular weight polypeptide (LMP7) was cloned and sequenced from a flounder (Paralichthys olivaceus) leukocyte cDNA library. The cDNA clone was 1,160 bp, and composed of an open reading frame of 822 bp that corresponded to a protein of 273 amino acid residues with a calculated mass of 30.5 kDa. The ScanProsite search indicated that the deduced amino acid sequence from the flounder LMP7 contains a proteasome beta-type subunit signature, which is well conserved during evolution. The sequence shares a high degree of identity with other LMP7 sequences varying from a 66% identity with zebra fish (Danio renio) to a 57% identity with the African clawed frog (Xenopus laevis), which was confirmed from a phylogenetic tree. A reverse transcription-polymerase chain reaction (RT-PCR) was used to determine tissue specificity, and the expression of LMP7 was detected from the liver, kidney, leukocyte, and spleen of the flounder.

Polymorphism of the angiotensin-converting enzyme gene in patients with cerebral infarction in Koreans.

The relationship between cerebrovascular disease and an insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme (ACE) gene is still being debated. The frequency of the DD genotype of the ACE gene was significantly higher in subjects with than those without cerebral infarction in Japan. The aim of the present study was to assess the relationship between ACE gene polymorphism and the development of cerebral infarction in a population from Korea. We examined its possible role as a risk factor in patients with cerebral infarction. The association between ACE gene polymorphism and cerebral infarction was examined in 106 patients with cerebral infarction and 498 controls without cerebral infarction. Frequencies of the genotypes and alleles of the ACE gene were investigated. The ACE genotype was analyzed by the polymerase chain reaction (PCR). The frequency of D allele was 37.7% in patients and 39.1% in controls (chi2 = 0.128, p = 0.720). The frequencies of the genotypes of the ACE gene were II: 39.6%, ID: 45.3%, and DD: 15.1% in patients, and II: 37.1%, ID: 47.6%, and DD: 15.3% in controls (chi2 = 0.127, p = 0.721). There was no significant difference in the frequency of the DD genotype of the ACE gene, and we did not find any association between ACE polymorphism and cerebral infarction. These results indicate that ACE polymorphism is not a risk factor for the development of cerebral infarction in a Korean population.

Inhibition of tumor necrosis factor-alpha-induced apoptosis by Asparagus cochinchinensis in Hep G2 cells.

A human hepatoma cell line, Hep G2 cells, is a reliable system for the study of alcohol-induced hepatotoxicity. In this study, we investigated the effect of an aqueous extract of Asparagus cochinchinensis(MERRIL) (Liliaceae) roots (ACAE) on ethanol (EtOH)-induced cytotoxicity in Hep G2 cells. ACAE (1-100 microg/ml) dose-dependently inhibited the EtOH-induced tumor necrosis factor-alpha (TNF-alpha) secretion. ACAE (1-100 microg/ml) also inhibited the EtOH and TNF-alpha-induced cytotoxicity. Furthermore, we found that ACAE inhibited the TNF-alpha-induced apoptosis of Hep G2 cells. These results suggest that ACAE may prevent the EtOH-induced cytotoxicity through inhibition of the apoptosis of Hep G2 cells.

Sonchus yellow net rhabdovirus nuclear viroplasms contain polymerase-associated proteins.

We have initiated a study of the cytopathology of nucleorhabdoviruses by analyzing the subcellular localization of sonchus yellow net virus (SYNV) genomic and antigenomic RNAs and the encoded polymerase proteins. In situ hybridizations demonstrated that the minus-strand genomic RNA sequences are restricted to the nuclei of infected cells, while the complementary plus-strand antigenomic RNA sequences are present in both the nuclei and the cytoplasm. Immunofluorescence and immunogold labeling experiments also revealed that the nucleocapsid (N) protein and phosphoprotein (M2) are primarily localized to discrete regions within the nuclei and in virus particles that accumulate in perinuclear spaces. The N protein antiserum specifically labeled the nuclear viroplasms, whereas the M2 antiserum was more generally distributed throughout the nuclei. Antibody detection also indicated that the polymerase (L) protein is present in small amounts in the viroplasm. When the N and M2 proteins were expressed individually from the heterologous potato virus X (PVX) vector, both proteins preferentially accumulated in the nuclei. In addition, viroplasm-like inclusions formed in the nuclei of cells infected with the PVX vector containing the N gene. Fusions of the carboxy terminus of beta-glucuronidase to N and M2 resulted in staining of the nuclei of infected cells following expression from the PVX vector. Deletion analyses suggested that multiple regions of the N protein contain signals that are important for nuclear localization.

Extraction of nuclei from sonchus yellow net rhabdovirus-infected plants yields a polymerase that synthesizes viral mRNAs and polyadenylated plus-strand leader RNA.

Although the primary sequence of the genome of the plant rhabdovirus sonchus yellow net virus (SYNV) has been determined, little is known about the composition of the viral polymerase or the mechanics of viral transcription and replication. In this paper, we report the partial isolation and characterization of an active SYNV polymerase from nuclei of SYNV-infected leaf tissue. A salt extraction procedure is shown to be an effective purification step for recovery of the polymerase from the nuclei. Full-length, polyadenylated SYNV N and M2 mRNAs and plus-strand leader RNA are among the products of the in vitro polymerase reactions. Polyadenylation of the plus-strand leader RNA in vitro is shown with RNase H and specific oligonucleotides. This is the first report of a polyadenylated plus-strand leader RNA for a minus-strand RNA virus, a feature that may reflect adaptation of SYNV to replication in the nucleus. Analysis of the SYNV proteins present in the polymerase extract suggests that the N, M2, and L proteins are components of the transcription complex. Overall, the system we developed promises to be useful for an in-depth characterization of the mechanics of SYNV RNA synthesis.

An RNA-dependent RNA polymerase activity associated with virions of tomato spotted wilt virus, a plant- and insect-infecting bunyavirus.

An RNA-dependent RNA polymerase activity was found associated with virions of tomato spotted wilt virus (TSWV), a plant- and insect-infecting member of the family Bunyaviridae. Radiolabeled nucleoside triphosphates were incorporated into trichloroacetic acid-precipitable products by detergent-disrupted, purified TSWV virions. Incorporation was reduced to near-background levels when RNase was present in the reaction mixture. The predominantly double-stranded RNA products were RNase-resistant at high but not low salt concentrations. The activity required manganese and was independent of a DNA template. Discrete products of approximately 3.0 kb and heterogeneous smaller products were synthesized that hybridized to purified TSWV RNA and transcripts of cDNA clones encompassing parts of each of the three genomic RNAs. The predominant products were viral sense although significant amounts of viral complementary sense S RNA products were also synthesized.

Sequence analysis of the trailer region of sonchus yellow net virus genomic RNA.

The sequence of the 5' terminus (or the "trailer" region) of the minus-sense RNA genome of sonchus yellow net virus (SYNV) was determined by dideoxynucleotide termination sequencing of purified viral RNA and cloned cDNAs. The 5'-terminal nucleotide was identified by nuclease P1 digestion of 32P-end-labeled genomic RNA followed by polyethyleneimine cellulose chromatography. The trailer sequence occupies positions 13,561 to 13,720 relative to the 3' end of the genomic RNA and is composed of 160 nucleotides (nt) adjacent to a dinucleotide forming a portion of the "gene junction" sequence at the terminus of the L protein gene. The trailer sequence is longer than the 144-nt plus-strand leader RNA transcribed from the 3' end of the genomic RNA and is the longest trailer sequence yet reported among the nonsegmented negative-strand viruses. As is characteristic of other rhabdovirus genomes, the 3' and 5' termini of the SYNV genome are complementary and are capable of forming a panhandle structure involving 16 of the 18 terminal nucleotides. However, there is no obvious direct nucleotide sequence relatedness between the SYNV trailer sequence and those of animal rhabdoviruses and paramyxoviruses. The existence of a minus-strand leader RNA of the same polarity as the trailer sequence could not be detected in nucleic acid extracted from infected plants under hybridization conditions suitable for detection of the plus-strand leader RNA. In this regard, SYNV differs from vesicular stomatitis virus and is similar to other rhabdoviruses which also fail to accumulate detectable minus-strand leader RNAs.