Evidence of multiple retrotransposons in two litopenaeid species.

Retrotransposons encompass a specific class of mobile genetic elements that are widespread across eukaryotic genomes. The impact of the varied types of retrotransposons on these genomes is just beginning to be deciphered. In a step towards understanding their role in litopenaeid shrimp, we have herein identified nine non-LTR retrotransposons, among which several appear to exist outside the standard defined clades. Two Litopenaeus stylirostris elements were discovered through degenerate PCR amplification using previously defined non-LTR degenerate primers, and through primers designed from a RAPD-derived sequence. A third genomic L. stylirostris element was identified using specific priming from an amplification protocol. These three PCR-derived sequences showed conserved domains of the non-LTR reverse transcriptase gene. In silico searching of genome databases and subsequent contig construction yielded six non-LTR retrotransposons (both genomic and expressed) in the Litopenaeus vannamei genome that also exhibited the highly conserved domains found in our PCR-derived sequences. Phylogenetic placement among representatives from all non-LTR clades showed a possibly novel monophyletic group that included five of our nine sequences. This group, which included elements from both L. stylirostris and L. vannamei, appeared most closely related to the highly active RTE clade. Our remaining four sequences placed in the CR1 and I clades of retrotransposons, with one showing strong similarity to ancient Penelope elements. This research describes three newly discovered retrotransposons in the L. stylirostris genome. Phylogenetic analysis clusters these in a monophyletic grouping with retrotransposons previously described from two closely related species, L. vannamei and Penaeus monodon.

A homozygous deletion on chromosome 3 in a small cell lung cancer cell line correlates with a region of tumor suppressor activity.

Small cell lung cancer (SCLC) tumors frequently display deletions on the short arm of chromosome 3 suggesting the existence of a 'tumor suppressor' gene within that region whose functional inactivation may be involved in tumorigenesis. Recently, a hybrid, HA(3)BB9F, was identified that contains a small fragment of human chromosome 3 of approximately 2 Mb on a mouse (A9) background (Killary et. al., 1992). This hybrid was utilized to define a functional tumor suppressor gene within 3p22-p21 which could suppress the tumorigenic properties of the mouse fibrosarcoma cell line. The existence of a tumor suppressor gene in the region 3p22-p21 is supported by the present report which describes the assessment of 89 SCLC and 32 non-SCLC lung cancer tumors and cell lines for the existence of a homozygous deletion(s) at 43 loci on the short arm of chromosome 3. One of the SCLC cell lines was found to harbor a homozygous deletion involving the loss of five markers on chromosome 3p. All five of the markers map to the region 3p21.3-p21.2 and four of the five markers are located within the chromosome 3 fragment exhibiting properties of tumor suppression in the HA(3)BB9F hybrid. The other tumors analysed all retained at least one copy of each of the markers assessed.

An 80 Kb P1 clone from chromosome 3p21.3 suppresses tumor growth in vivo.

High frequencies of allelic loss on the short arm of chromosome 3 in small cell lung cancer (SCLC) and a number of other tumors suggest the existence of a tumor suppressor gene(s) within the deleted regions. Two small cell lung cancer lines, NCI H740 and GLC20, have been described which have homozygous deletions in the region 3p21.3. The deleted region overlaps with a 2 Mb fragment of human DNA present in the interspecies hybrid HA(3)BB9F, that suppresses tumor formation by mouse A9 fibrosarcoma cells. Human sequences from this cell hybrid were isolated using inter Alu PCR. From this starting point, a P1 contig was developed for the region of 450 Kb that is common to the homozygous deletions seen in the SCLC lines NCI H740 and GLC20 and is also present in HA(3)BB9F, the suppressed A9 hybrid. Individual P1 clones were assayed for their ability to suppress the tumorigenicity of the mouse fibrosarcoma cell line A9 as assayed by injection of transfected A9 cells into athymic nude mice. The introduction of one of the P1 clones into A9 cells resulted in suppression of tumor growth whereas two other P1 clones from the contig failed to suppress tumor formation in athymic nude mice. These data functionally delimit a tumor suppressor locus to a region of 80 kb within a P1 clone at 3p21.3.

Elevation of a specific mRNA in longissimus muscle of steers fed ractopamine.

To address the hypothesis that some classes of growth promoters stimulate muscle protein synthesis in growing cattle, 23 crossbred steers were fed diets containing the phenethanolamine growth promoter ractopamine in a 140-d feeding trial. Steers received either no ractopamine, .18 or .36 mg.kg BW-1.d-1 ractopamine for 140 d or .36 or .72 mg.kg BW-1.d-1 ractopamine for 56 d. Longissimus muscle was obtained at slaughter and frozen in liquid N2. RNA was extracted by homogenization of pulverized frozen muscle in guanidinium isothiocyanate and centrifugation through cesium chloride. Polyadenylated mRNA was extracted by capture on oligo-dT columns. Ractopamine had no effect on total RNA or mRNA concentrations (P greater than .25). Hybridization of the RNA to a putative myosin light chain-1/3 (MLC-1/3) cDNA clone in a Northern blot indicated one heavy band (approximately 1 kb) with no evidence of extensive destruction of the RNA. A second, minor band (approximately 3 kb) also was observed in some samples. The MLC-1/3 cDNA clone was hybridized to 1- or 5-micrograms samples of total RNA, and the intensity of the resultant autoradiographs was quantified by laser densitometry. There was a statistical correlation between MLC-1/3 mRNA-micrograms RNA-1 and longissimus cross-sectional area (P less than .05) and average daily gain (P less than .025). The results suggest that ractopamine either increased the transcription of the putative MLC-1/3 gene and(or) increased the stability of MLC-1/3 mRNA in bovine longissimus muscle, either of which could result in an increase in specific myofibrillar protein synthesis.

Adipose tissue, longissimus muscle and anterior pituitary growth and function in clenbuterol-fed heifers.

The present study was conducted to determine the effects of feeding clenbuterol on adipose tissue and longissimus muscle growth in heifers. For 50 d, 14 heifers were fed either a sucrose-based, clenbuterol supplement or a placebo in which the clenbuterol had been omitted. The heifers were slaughtered in two groups, based on initial weight. Adipose tissue from several anatomical sites and longissimus muscle (depending on slaughter group) were obtained fresh at slaughter. Changes in carcass characteristics elicited by clenbuterol were similar to those reported by others for steers and sheep. Subcutaneous (sc) and intramuscular (im), but not perirenal, adipocytes were smaller and there were more cells per g tissue in the adipose tissue depots of the clenbuterol-fed heifers. Clenbuterol decreased lipogenic enzyme activities, fatty acid-binding protein activity, basal lipolysis and acetate incorporation into glyceride-fatty acids (P less than .05) in sc adipose tissue, but had no effect (P greater than .05) on lipogenesis or lipolysis in im adipose tissue. Clenbuterol elicited a 20% increase in type II myofiber diameters (P less than .05) but had no effect on type I myofiber diameters. In vitro growth hormone release by perifused anterior pituitaries was not affected significantly by long-term in vivo exposure to clenbuterol. These data indicate that a depression in lipogenesis is the mechanism by which clenbuterol decreases subcutaneous fat accretion in cattle.

Molecular analysis of a RAPD marker (B20) reveals two microsatellites and differential mRNA expression in Penaeus vannamei.

We previously reported a population-specific DNA fragment (B20) in Penaeus vannamei shrimp, fragment found using the randomly amplified polymorphic DNA (RAPD) procedure, that was present in Population 2 but not in Populations 1 and 4. The specific objectives of this study were to clone and sequence this genetic marker, determine if all or part of this cloned sequence could be found in any of the other populations in which this marker could not be amplified, and examine if this marker represents a functional gene by examining the steady-state levels of mRNA expression using Northern blot hybridization. Sequence information of the 1259-bp B20 clone revealed two microsatellites and two candidate open reading frames. Although the entire B20 sequence could only be amplified in Population 2 (from Ecuador), Population 3 (a hybrid of Populations 1 and 2), and a few individuals from wild Ecuadorian shrimp samples, portions of the B20 DNA could be amplified in individuals from Populations 1, 2, 3, candidate Population 4, and wild Ecuadorian samples. These microsatellites vary in size between populations and families. Northern blot hybridization analysis using radiolabeled B20 probe detected two mRNA transcripts of approximately 1.5 and 2.0 kb. Expression data throughout development indicated that these transcripts were present at low levels in nauplii from two of the three crosses examined using broodstocks of Population 1. Higher levels were observed in postlarvae (PL) 6, PL8, and PL10 in one of the three crosses. Individuals from all crosses showed higher levels of expression in the juvenile tail muscle. The mRNA transcript levels were undetected in zoea 3, PL2, and PL4 stages of development and broodstock tail muscle. The levels of expression of B20 mRNA transcripts varied significantly between Populations 1, 2, 3, 4, and wild Ecuadorian individuals as well as between families and within individuals representative of seven families from Population 1. In summary, the B20 clone revealed the presence of two microsatellites that vary in size between populations. These microsatellites will be useful for estimating genetic diversity within and between populations, identifying family-specific markers, and mapping loci responsible for economically important traits in penaeid shrimp. The mRNA levels detected by the B20 clone showed differential expression during development, and the pattern of expression was influenced by the genetic background of the parental crosses used.

Genetic markers applied in regression tree prediction models.

Classification and regression tree (CART) modelling was used to determine infectious hypodermal and haematopoietic necrosis virus (IHHNV) resistance and susceptibility in Penaeus stylirostris. In a previous study, eight random amplified polymorphic DNA (RAPD) markers and viral load values using real-time quantitative PCR were obtained and used as the training data set in order to create numerous regression tree models. Specifically, the genetic markers were used as categorical predictor variables and viral load values as the dependent response variable. To determine which model has the highest predictive accuracy for future samples, RAPD fingerprint data was generated from new Penaues stylirostris IHHNV resistant and susceptible individuals and used to test the regression models. The best performing tree was a four terminal node tree with three genetic markers as significant variables. Marker-assisted breeding practices may benefit from the creation of regression tree models that apply genetic markers as predictive factors. To our knowledge this is the first study to use RAPD markers as predictors within a CART prediction model to determine viral susceptibility.

Nucleotide sequence of 3'-end of the genome of Taura syndrome virus of shrimp suggests that it is related to insect picornaviruses.

Taura syndrome disease, caused by Taura syndrome virus (TSV), is one of the most important viral diseases of penaeid shrimp in the Western Hemisphere resulting in catastrophic disease epidemics in farmed shrimp. We have cloned and sequenced a 3278 bp cDNA representing the 3' end of the TSV genome. Sequence analyses revealed that frame + 2 had the longest open reading (ORF) frame. This frame contained a 5'-terminal 19 non-coding bases followed by an ORF from nucleotides 20 to 3053 (encoding 1011 amino acids, aa) and a 3' untranslated region of 225 nts. The deduced aa sequence of TSV showed significant similarities with those of the coat proteins of insect picornaviruses, Rhopalosiphum padi virus, Plautia stali intestine virus, Drosophila C virus, Triatoma virus of Triatoma infestans and Himetobi P virus of brown plant hopper. A single transcript of approximately 10 kb was detected by Northern blot hybridization suggesting that the TSV coat protein gene is not expressed as a subgenomic RNA. We concluded that the genome organization of TSV is similar to insect picornaviruses. This is the first molecular evidence of occurrence of a picornavirus in the class Decapoda.

Genetic diversity of cultured Penaeus vannamei shrimp using three molecular genetic techniques.

Three molecular genetic techniques, restriction fragment length polymorphisms (RFLPs), random amplification of polymorphic DNA (RAPD), and allozyme variability, were used to evaluate the genetic diversity of two specific-pathogen-free (SPF) populations (numbers 1 and 2) and one candidate SPF population (number 4) of Penaeus vannamei developed and maintained by the U.S. Marine Shrimp Farming Program. A total of 114 individuals were tested, which included 30 each from families 1.5 and 1.6 of population 1 and from population 2, and 24 from population 4. Two HhaI mitochondrial DNA polymorphisms (A and B) were found in all the animals examined, with family 1.5 and population 2 showing type A and family 1.6 showing type B. After scoring 73 bands obtained with six different RAPD primers, the percentage of polymorphic bands was: 55% for families 1.5 and 1.6 of population 1, 48% for population 2, and 77% for population 4, suggesting that population 4 is the most polymorphic of all three populations. The allozymic variation at 30 loci showed no fixed differences in isozyme genotypes between families 1.5 and 1.6. The percentage of polymorphic loci, under the criterion that the frequency of the most common allele was less than 0.95 in each population, was 6.67%, 3.33% and 16.67% for family 1.5 of population 1, family 1.6 of population 1, and population 2, respectively. Mean heterozygosities (+/- SE) were 0.023 +/- 0.017, 0.018 +/- 0.016, and 0.064 +/- 0.026, respectively. The low levels of allozyme polymorphisms indicate that mitochondrial DNA and nuclear DNA techniques are more useful for examining genetic diversity in order to follow individual stocks within a breeding program and to correlate genotypes with desirable growth and reproductive performance of SPF P. vannamei stocks.

Bovine fast-twitch myosin light chain 1: cloning and mRNA amount in muscle of cattle treated with clenbuterol.

The cDNA clone encoding the fast-twitch isoform of myosin light chain 1 (MLC-1f) was isolated from bovine longissimus dorsi muscle and sequenced in M13 and pUC8. An 0.8-kb subclone, produced by digestion of the cDNA with EcoRI, contained the portion of the molecule common to MLC-1f and MLC-3f. The cDNA in pUC8 contained an additional 81 bp upstream of the EcoR I digestion site, which was unique to MLC-1f. The cDNA clone was used to measure MLC-1f mRNA in longissimus dorsi muscle of cattle chronically administered the beta-adrenergic agonist clenbuterol. Treatment with clenbuterol for 50 days increased succinic dehydrogenase negative (type IIB) and positive (types I and IIA) myofiber cross-sectional areas by 25%. After the 50-day treatment period, the amount of MLC-1f mRNA was 90% greater in longissimus dorsi muscle of treated animals than in the initial group. This effect was lost when clenbuterol treatment was withdrawn for a 78-day period, during which time muscle growth in the treated animals stopped completely. We conclude that we have cloned the bovine cDNA for MLC-1f, which has provided additional evidence that beta-adrenergic agonists increase myofibrillar gene expression.

Genetic structure and chromosomal mapping of MyD88.

The myeloid differentiation (MyD) marker MyD88 was initially characterized as a primary response gene, upregulated in mouse M1 myeloleukemic cells in response to differentiation induced by interleukin-6. Subsequent analysis revealed that MyD88 possesses a unique modular structure, which consists of an N-terminal "death domain," similar to the intracellular segments of TNF receptor 1 and Fas, and a C-terminal region related to the cytoplasmic domains of the Drosophila morphogen Toll and vertebrate interleukin-1 receptors. In this report we describe the cloning and gene structure of mouse MyD88. The complete coding sequence of mouse MyD88 spans five exons, with the first exon encoding the complete death domain. Zooblot analysis revealed that MyD88 is an evolutionarily conserved gene. MyD88 was localized to the distal region of mouse chromosome 9 by interspecific backcross mapping. The human homolog (hMyD88) was mapped to chromosome 3p22-p21.3 by PCR analysis of a human chromosome 3 somatic cell hybrid mapping panel. Northern blot analysis revealed widespread expression of MyD88 in many adult mouse tissues, and RT-PCR studies detected MyD88 mRNA in T and B cell lines and differentiating embryonic stem cells. The broad expression pattern demonstrates that mouse MyD88 expression is not restricted to cells of myeloid lineage as was originally believed.

Isolation of the human semaphorin III/F gene (SEMA3F) at chromosome 3p21, a region deleted in lung cancer.

Small cell lung cancer (SCLC) has been correlated with a deletion in the short arm of chromosome 3, with the region 3p21 being lost from one homolog in almost all cases. Two SCLC cell lines have homozygous deletions in 3p21, and these deletions overlap with a fragment of chromosome 3 that has tumor suppression activity in vivo. We have isolated some cDNA clones from this region that are homologous to the genes constituting the semaphorin family. They represent a novel human semaphorin, termed sema III/F (HGMW-approved symbol SEMA3F), which is expressed as a 3.8-kb transcript in a variety of cell lines and tissues; it is detected as early as Embryonic Day 10 in mouse development. There is high expression in mammary gland, kidney, fetal brain, and lung and lower expression in heart and liver. Although there is reduced expression of this gene in several SCLC lines, no mutations were found. This semaphorin homolog has characteristics of a secreted member of the semaphorin III family, with 52% identity with mouse semaphorin E and 49% identity with chicken collapsin/semaphorin D.

Localization of the Fanconi anemia complementation group D gene to a 200-kb region on chromosome 3p25.3.

Fanconi anemia (FA) is a rare autosomal recessive disease manifested by bone-marrow failure and an elevated incidence of cancer. Cells taken from patients exhibit spontaneous chromosomal breaks and rearrangements. These breaks and rearrangements are greatly elevated by treatment of FA cells with the use of DNA cross-linking agents. The FA complementation group D gene (FANCD) has previously been localized to chromosome 3p22-26, by use of microcell-mediated chromosome transfer. Here we describe the use of noncomplemented microcell hybrids to identify small overlapping deletions that narrow the FANCD critical region. A 1.2-Mb bacterial-artificial-chromosome (BAC)/P1 contig was constructed, bounded by the marker D3S3691 distally and by the gene ATP2B2 proximally. The contig contains at least 36 genes, including the oxytocin receptor (OXTR), hOGG1, the von Hippel-Lindau tumor-suppressor gene (VHL), and IRAK-2. Both hOGG1 and IRAK-2 were excluded as candidates for FANCD. BACs were then used as probes for FISH analyses, to map the extent of the deletions in four of the noncomplemented microcell hybrid cell lines. A narrow region of common overlapping deletions limits the FANCD critical region to approximately 200 kb. The three candidate genes in this region are TIGR-A004X28, SGC34603, and AA609512.