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PubMed1994

Rapid chemiluminescent detection of the mRNA for uncoupling protein in brown adipose tissue by northern hybridization with a 32-mer oligonucleotide end-labelled with digoxigenin.

Sequences for the uncoupling protein gene (or mRNA) expressed in brown adipose tissue have been re-examined for homology between species. A 32 bp region is identical in rats and cattle, this representing a 5 bp extension of the 27 bp conserved sequence previously described. In mice, rabbits, Syrian hamsters, and humans there is a single bp difference with the 32 bp conserved rat/cattle sequence. A 32-mer antisense oligonucleotide (3'-TAGTG-GAAGGGCGACCTGTGGCGGTTTCAGGC-5') targeted to the 32 bp rat/cattle sequence has been synthesized and end-labelled (3' and 5' ends) with digoxigenin, as a simple, non-radioactive probe for uncoupling protein mRNA. Hybridization of the oligonucleotide to the mRNA was readily detected on Northern blots with total RNA from brown adipose tissue of rats, mice, and newborn lambs, using a chemiluminescence substrate and following short exposure to film (1 h, or less). With longer exposure (overnight), uncoupling protein mRNA was detected in as little as 250 ng of total RNA from rat brown fat. The 32-mer digoxigenin-labelled oligonucleotide, coupled with a chemiluminescence substrate, provides a rapid, sensitive, non-radioactive procedure for detecting uncoupling protein mRNA on Northern blots, applicable across species.

Adipose Tissue, Brown↗
PubMed1990

cDNA library construction from small amounts of unfractionated RNA: association of cDNA synthesis with polymerase chain reaction amplification.

We describe here a general and simple procedure for cDNA library construction making use of in vitro amplification of cDNA by polymerase chain reaction (PCR). The first-strand cDNA is synthesized from total RNA with a primer EcoRI-(dT)17 and oligo(dG) tailed. An oligonucleotide, EcoRI-BamHI-(dC)13, is used to prime the second-strand synthesis by the thermostable DNA polymerase of Thermus aquaticus. The double-stranded cDNA is then amplified directly by PCR. A study of the effect of the elongation time on the PCR products showed that a long extension time is necessary to overcome the size heterogeneity of the cDNA population. Starting from 1 microgram of total brain RNA, the products obtained ranged from 200 to more than 2000 bp. The presence of the myelin basic protein cDNA sequence was determined. A lambda gt10 library containing 2 x 10(6) clones was established with the amplified cDNA. No sequences originating from rRNA were detected by Southern blot analysis. The ability to produce representative cDNA libraries from minute amounts of total RNA by this protocol should have many applications to studies of gene expression in small amounts of tissues or cells.

Animals↗
PubMed1985

Bleomycin selectively elevates mRNA levels for procollagen and fibronectin following acute lung injury.

We employed the technique of dot blot hydridization of radiolabeled cDNA probes to examine the role of specific mRNA content in the control of extracellular matrix turnover in the remodeling rat lung. Following bleomycin instillation, total RNA content gradually doubled during the first 5 days following the initial lung injury, then rose much more rapidly during the ensuing 9 days. Individual mRNAs for procollagens I and III and for fibronectin were selectively enriched 2- to 4-fold above total RNA during the first week after bleomycin instillation. No comparable increases were observed in specific RNAs from liver, indicating that the response observed in the lung was not generalized to other organs. Moreover, the increases in mRNA species for procollagen types I or III in the lung could not be related to the influx of inflammatory cells which migrate into the lungs during acute injury, as cells obtained by alveolar lavage contained no mRNAs for procollagens.

Animals↗
PubMed1985

Comparisons of human placental lactogen mRNA levels from placentas of diabetics and normal term.

Overt diabetes and gestational diabetes (1-2.5% of all pregnancies) has been related to perinatal mortality, increased macrosomia and increased frequency of other pregnancy complications. Human placental lactogen (hPL), a hormone similar to growth hormone, is produced by the placenta and is a potent antagonist to insulin action. While hPL's presence in maternal circulation induces a sparing effect on nutrients including glucose, it exacerbates diabetes during pregnancy and may well relate to other clinical complications. To explore possible regulation of hPL in diabetic pregnancy and specifically to examine gestational diabetes, we have evaluated the levels of placental mRNA coding for hPL synthesis as well as other parameters from diabetic and normal term patients. By in vitro translation assays using nuclease-treated reticulocyte lysate, no substantial differences in translatable hPL-mRNA were observed when comparing normal term (3.5% of total synthesis), gestational diabetic (3.4%) and Type C diabetic (3.5%). However, translatable hPL-mRNA in Type R diabetes which was 2.7% of total synthesis was slightly reduced in comparison to normal term. To determine more directly hPL-mRNA levels in gestational diabetic placentas and normal term placentas, total RNA preparations were evaluated qualitatively by northern blot and quantitatively by dot blot of RNA and cDNA hybridization to a nick-translated hPL-pMB9 plasmid. The northern blot revealed no major size differences of the mRNA and the dot blot hybridization was quantitatively similar for both gestational diabetics and normal terms per unit of total RNA. By direct analysis of DNA per g tissue we found the DNA content of placentas from gestational diabetics and normal term to be statistically the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Female↗
PubMed1997

Two alleles of the human paraoxonase gene produce different amounts of mRNA. An explanation for differences in serum concentrations of paraoxonase associated with the (Leu-Met54) polymorphism.

In a recent study we demonstrated that a polymorphism of the human paraoxonase gene affecting position 54 was linked to variations in serum concentrations of the enzyme. L allele carriers (leucine at position 54) have significantly higher concentrations of paraoxonase than M allele carriers (methionine at position 54). In the present study we examined the hypothesis that differences in mRNA production could contribute to variations in serum concentrations. Relative concentrations of L and M type mRNA were analyzed in total RNA extracted from heterozygous liver samples. This was achieved by cDNA synthesis, polymerase chain reaction amplification of the cDNA fragment containing the 54 polymorphism and restriction analysis to identify radiolabeled end fragments of L and M alleles. An allele mixing experiment using total RNA from liver samples of LL and MM homozygotes demonstrated the sensitivity of the approach to changes in the relative concentrations of each type of RNA. In 8 of 10 heterozygous samples, an excess of L allele type mRNA was observed. Overall there was a significantly higher level of L type mRNA (L:M ratio of 2.51 +/- 1.41, n = 10, P < .01). These results support our hypothesis that increased concentrations of serum paraoxonase arise from greater production of L allele mRNA. In two samples, the L:M ratio was close to or below 1.0. This is consistent with the known spectrum of paraoxonase serum concentrations associated with the L and M alleles and suggests that factor(s) that preferentially modulate allele expression are usually, but not uniformly, associated with the L allele.

Alleles↗
PubMed1987

Developmental regulation of somatostatin gene expression in the brain is region specific.

Developmental regulation of somatostatin (SRIF) gene expression was studied in five regions of rat brain and in rat stomach. Total RNA was isolated from hypothalamus, cortex, brainstem, cerebellum, and olfactory bulb, as well as stomach at eight stages of development from prenatal day 16 to postnatal day 82. Hybridization of a 32P-labeled rat SRIF cDNA probe to Northern blots of total RNA from the above tissues during development demonstrated a single hybridizing band approximately 670 base pairs in length. When SRIF mRNA levels from each stage of development were quantified and normalized by the amount of poly (A)+ RNA present at that stage of development, a unique pattern of SRIF gene expression was seen in each region. In brainstem and cerebellum, SRIF mRNA levels peaked early in development between prenatal day 21 and postnatal day 8 and then declined until postnatal day 82. Hypothalamus and cortex, on the other hand, showed a progressive increase during development with peak levels occurring between postnatal days 13 and 82. In contrast, stomach and olfactory bulb showed SRIF mRNA levels which were low during early development and which rose late in development (postnatal days 13 to 82). Marked differences in the amount of SRIF mRNA within each region were present as well. These data suggest that there is differential expression of the SRIF gene in different regions of the brain and in the stomach during development. Further study of this phenomenon may provide insight into the in vivo control of SRIF gene expression and the role of SRIF in the developing brain.

Animals↗
PubMed1984

Rat brain preproenkephalin mRNA. cDNA cloning, primary structure, and distribution in the central nervous system.

A cDNA-clone library was constructed from poly-adenylated RNA of Fischer rat striatum and screened for inserts coding for the enkephalin precursor preproenkephalin. The insert of one positive clone, pRPE2, was sequenced and found to contain the coding sequence (810 bases), as well as 316 and 155 bases of the 3' and 5' untranslated regions, respectively, of rat preproenkephalin mRNA. The primary structure of rat preproenkephalin (269 amino acids, Mr 30,932) is similar to those of previously sequenced bovine and human preproenkephalins (78 and 82% matched residues, respectively), and contains four copies of Met-enkephalin, one of Leu-enkephalin, one of Met-enkephalin-Arg6-Gly7-Leu8, and one of Met-enkephalin-Arg6-Phe7. Cell-free translation of rat striatal mRNA selected by hybridization with pRPE2 DNA resulted in the synthesis of a 31,000-Da protein. Southern analysis of rat genomic DNA with 32P-labeled pRPE2 fragments is consistent with a single preproenkephalin gene. A 32P-labeled pRPE2 fragment hybridized specifically with preproenkephalin mRNA (approximately 1500 bases) on Northern blots of polyadenylated or total RNA of all brain regions but not liver. Relative abundances of preproenkephalin mRNA in total RNA of specific regions of the rat central nervous system, determined by a sensitive dot-blot hybridization assay, had the following order: striatum much greater than hypothalamus, pons-medulla, spinal cord greater than cerebellum, midbrain, frontal cortex greater than hippocampus, thalamus. The pRPE2 probe is useful for the analysis of the dynamics of preproenkephalin mRNA in rat neurons.

Amino Acid Sequence↗
PubMed2002

Stabilization of RNA strands in protein synthesis by type I procollagen C-proteinase enhancer protein, a potential RNA-binding protein, in hepatic stellate cells.

Type I procollagen C-proteinase enhancer (PCPE) exists in hepatic stellate cells (HSCs) which can produce collagen. The deduced amino acid sequence of PCPE contains motifs specific for RNA-binding proteins. The effect of PCPE on the syntheses of collagen and noncollagenous protein was studied using an HSC clone derived from cirrhotic rat liver. When the cells were cultured in the presence of an antisense oligonucleotide (AS) against PCPE mRNA, the synthesis of noncollagenous protein as well as collagen was reduced compared to the cells cultured with addition of a nonsense oligonucleotide (NS). The extent of the reduction was similar in both syntheses. The total RNA content of the AS-treated cells and NS-treated cells did not differ. In the presence of actinomycin D, however, such total RNA content was decreased more rapidly in the AS-treated cells than in the NS-treated cells. PCPE may be involved in stabilization of RNA strands in noncollagenous protein synthesis as well as collagen synthesis.

Adipocytes↗
PubMed1987

Hormonal regulation of RNA synthesis and specific gene expression in Xenopus oviduct cells in primary culture.

The studies described in this report were carried out as a first step towards the elucidation of mechanisms underlying the tissue specificity of regulation of gene expression by estrogen. Using a procedure established earlier in our laboratory for primary culture of Xenopus hepatocytes, we have characterized how estradiol-17 beta and progesterone affect the rate of synthesis of total RNA and that of accumulation of two oviduct-specific mRNAs in Xenopus oviduct cells in primary culture. In cells that had recovered from 'culture shock' 3 days after they were plated out, both hormones had only a slight or no effect on the overall rate of labelling of newly synthesized RNA over 24 h. Cloned cDNA probes for two mRNAs, termed 7F and 6G and specifying as yet unknown proteins expressed in the oviduct and not in the liver, were used to quantify the two mRNAs. The levels of both mRNAs declined for the first 2 days in culture after which they were stabilized. When added to the oviduct cell cultures 3 days after they were plated out, estradiol increased the steady-state concentration (relative to total RNA) of 7F and 6G mRNAs by 3- to 7-fold after 60-80 h, but with different time-course and dose-response kinetics for the two messages. The antiestrogen tamoxifen also exerted different degrees of antagonist effect on the estrogen-induced accumulation of 7F and 6G mRNAs. Although the protein products of these two oviduct-specific mRNAs have not yet been characterized, these studies set the stage for comparing the regulation by estrogen of their genes with that of vitellogenin genes in primary cultures of Xenopus oviduct cells and hepatocytes.

Animals↗
PubMed1994

Characterisation of cDNA and genomic clones encoding homologues of the 65 kDa regulatory subunit of protein phosphatase 2A in Arabidopsis thaliana.

Two cDNA species encoding sequences homologous to the 65 kDa regulatory subunit (PR 65) of protein phosphatase 2A (PP2A) have been isolated from an Arabidopsis thaliana cDNA library. These were designated pDF1 and pDF2. pDF1 is 1795 bp long and by comparison with the human and porcine PP2A regulatory subunit sequences represents a full-length clone. It encodes a predicted polypeptide of 587 amino acid residues. pDF2 is truncated at the 5' end by 237 bp. The complete nucleotide sequences have been determined for both cDNA species. Comparison of the nucleotide and the deduced amino acid sequences showed that the two sequences were homologous but not identical and therefore must be derived from two different genes. Northern blot analysis was performed on total RNA and poly(A)+ RNA isolated from seed at various stages of development and from young leaf material of Brassica napus L. (oilseed rape). Both cDNA probes hybridised to a single major mRNA species of ca. 2.2 kb. The highest level of expression was observed in the total RNA from developing rape seed at about 33 days after flowering, and the transcript level in the poly(A)+ RNA of the seed was higher than in young leaf of oilseed rape. Southern blot analysis was performed on two varieties of A. thaliana and B. napus genomic DNA; this identified a small family of genes in A. thaliana consisting of at least 2 or 3 members and a larger multigene family in B. napus of at least 5 or 6 members. Two independent genomic clones were isolated from an A. thaliana genomic library. Sequencing of a fragment common to both revealed that the sequence was identical in both clones and, therefore, they were assumed to contain the same genomic sequence. The genomic sequence selected, designated regA, is 3639 bp long and the coding sequence contains eleven introns. The gene encodes a predicted polypeptide of 590 amino acid residues. The sequence comparison with both cDNA sequences showed that it is homologous but not identical to the two, confirming that at least three different genes exist in A. thaliana which encode PR65 of PP2A.

Amino Acid Sequence↗
PubMed1996

Different Effects of Lipopolysaccharide on Plasminogen Activator Inhibitor-1 Production in Aortic Media in Vivo and in Culture.

Background: Lipopolysaccharide (endotoxin) has been shown to increase the expression of plasminogen activator inhibitor type-1 (PAI-1) in the vessel wall. Endotoxin is known to increase PAI-1 production in endothelial cells, but its action on smooth muscle cells (SMCs) is presently not clear. In this study we determined the effect of endotoxin on PAI-1 and tissue plasminogen activator (t-PA) production by aortic SMCs in vivo in two animal species, and in culture. Methods: The aortas of Sprague Dawley rats and of New Zealand White rabbits were rapidly excised after parenteral administration of endotoxin. Total RNA was extracted from the aortic media, and PAI-1 and t-PA mRNA levels were quantified after Northern blotting. In addition, cultured rat aortic SMCs were treated with endotoxin. PAI activity in the conditioned medium was determined with a spectrophotometric assay, and total RNA was extracted from the cells and analyzed. Results: A rapid and strong induction in the aortic medi a of PAI-1 mRNA was observed by endotoxin in both rat (50 mg/kg) and rabbit (1 mg/kg). t-PA mRNA was barely detectable and was not increased by endotoxin. Studies in cultured SMCs showed low expression of PAI-1 mRNA under serum-free conditions and little PAI activity in the cell-conditioned medium. Endotoxin did not increase the levels of PAI-1 mRNA nor PAI activity under serum-free conditions. The effect of endotoxin (10 mg/ml) in the presence of 10% (v/v) newborn calf serum on PAI-1 mRNA was negligible; PAI activity, however, increased by 50.3 +/- 7.3% compared with controls. mRNA levels of t-PA and low-density lipoprotein/receptor-related protein/alpha2-macroglobulin receptor also increased after endotoxin administration. PAI activity was identified as PAI-1 by immunoblotting. Fibrin zymography showed that t-PA was present only in complex with PAI-1. Conclusions: A strong increase in PAI-1 gene expression by endotoxin was observed in aortic SMCs in vivo but not in culture. Th is suggests that the effect of endotoxin on SMCs is indirect. The fibrinolytic/proteolytic potential of the SMCs in the vessel wall is likely to have important implications for the migration of cells during vessel wall remodeling, such as neointima formation, during tumor cell metastasis, and for the fate of intramural thrombi.

Journal Article↗
PubMed2000

Porcine erythropoietin receptor: molecular cloning and expression in embryonic and fetal liver.

The full coding sequence for porcine erythropoietin receptor (EPOR) was elucidated using reverse transcription polymerase chain reaction (PCR) (rtPCR) and 3' and 5' rapid amplification of cDNA ends (RACE) procedures. Total RNA collected from Day 30 fetal liver was used as starting material. A 1843 bp sequence was obtained from which could be inferred a 509 amino acid protein which was 79-85% identical to the amino acid sequence of erythropoietin receptor from other species. Total RNA samples collected from white crossbred intact, white crossbred UHO and Meishan gilts on Days 24, 30 and 40 of gestation were subjected to Northern blotting using porcine EPOR cDNA as probe. Results indicated that (1) a major and two minor forms of mRNA are present, (2) fetal liver mRNA concentrations for EPOR are low on Day 24 of gestation and increase dramatically by Day 30 and (3) mRNA concentrations for EPOR tended to be decreased by intrauterine crowding.

Amino Acid Sequence↗
PubMed1989

Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY).

The polymerase chain reaction (PCR) is used as part of a new approach to the absolute quantification of mRNA. We describe a PCR aided transcript titration assay (PATTY) which is based on the co-amplification of an in vitro generated transcript differing by a single base exchange from the target mRNA. Identical portions of a total RNA sample are "spiked" with different amounts of this mutated standard RNA, converted to cDNA and amplified by PCR. Because the base exchange creates a novel restriction endonuclease site, the ratio of co-amplified DNA derived from target mRNA to amplified DNA derived from standard RNA can be determined after restriction endonuclease digestion and separation by gel electrophoresis. This method gives accurate results within 24 hours and is useful especially for the quantification of either low-abundance mRNA or more abundant mRNA present in very small amounts of total RNA. The low-abundance mRNA encoding 4-coumarate:CoA ligase (4CL) in cultured potato cells (Solanum tuberosum L.) was measured in a case study. About 100 molecules per assay could be accurately detected by the new method.

Base Sequence↗
PubMed2006

Corticosterone impairs the mRNA expression and activity of 3beta- and 17beta-hydroxysteroid dehydrogenases in adult rat Leydig cells.

Clinical and experimental studies, including our own observations, have shown the adverse effects of excess glucocorticoids on testicular steroid hormone production. The present study was designed to gain insight into the molecular mechanisms by which excess corticosterone impairs Leydig cell steroidogenesis. To achieve this, adult rats were administered with corticosterone-21-acetate (2 mg/100 g body weight) twice daily for 15 days. After the treatment period, rats were killed by decapitation. The testes were removed, decapsulated aseptically and used for the isolation of Leydig cells. Purified Leydig cells were used for assessing the activity of 3beta- and 17beta-hydroxysteroid dehydrogenases (HSDs) and total RNA isolation. For in vitro studies, purified Leydig cells (7.5 x 10(6) cells) of control rats were plated in culture flasks and exposed to different concentrations (50, 100, 200, 400, and 800 nmol/L) of corticosterone for 24 h. At the end of incubation, total RNA was isolated from cultured Leydig cells, and the mRNA of 3beta- and 17beta-HSDs was quantified by RT-PCR. A significant reduction in the activities and levels of 3beta-HSD type-I and 17beta-HSD type-III mRNAs in Leydig cells were observed. In vitro studies demonstrated a dose-dependent significant impairment in both the activity and mRNA expression of these enzymes. These results suggest that corticosterone might have a direct effect on the transcription of the genes of 3beta- and 17beta-HSD. It is inferred from the present in vivo and in vitro studies that one of the molecular mechanisms by which excess corticosterone decreases the steroidogenic potency of Leydig cells is by suppressing the mRNA expression of 3beta-HSD type-I and 17beta-HSD type-III enzymes.

17-Hydroxysteroid Dehydrogenases↗
PubMed1994

Quantitation of interferon gamma mRNA levels in psoralen/UVA-treated HUT-78 cells by competitive PCR.

We have developed a method to accurately quantitate IFN gamma mRNA in HUT-78 cells before and after PUVA treatment by the competitive RT/PCR technique, which could be utilized to accurately quantitate any mRNA species of interest. Total RNA was isolated from HUT-78 cells before and after PUVA treatment. A synthetic IFN gamma mRNA was made to contain a 54 bp deletion in the middle of IFN gamma cDNA gene and used as an internal standard. 0.5 microgram of target RNA was co-reverse transcribed and co-amplified with increasing concentrations of synthetic IFN gamma RNA using the same primers. The products of the synthetic RNA were separated from that of the target RNA by gel electrophoresis. This allowed determination of the amount of target IFN gamma mRNA to be quantitated by extrapolating against a standard curve. PUVA treatment of HUT-78 cells resulted in an increase in IFN gamma mRNA level from 32 to 80 pg/microgram of total RNA, suggesting that PUVA induces transcription of T-helper 1 cytokines as part of its mechanism of action.

Base Sequence↗
PubMed2000

Effects of tumor necrosis factor-binding protein on hepatic protein synthesis during chronic sepsis.

BACKGROUND: Cytokines are thought to play a role in the stimulation of protein synthesis in liver during inflammation and sepsis. We previously showed that administration of tumor necrosis factor-binding protein (TNFbp) prevents the sepsis-induced inhibition of protein synthesis in skeletal muscle. The purpose of the present set of experiments was to investigate the effect of TNFbp on hepatic protein synthesis and its ability to modulate the mechanisms responsible for increased hepatic protein synthesis during chronic (5-day) intraabdominal sepsis. MATERIALS AND METHODS: We examined the effects of TNFbp on hepatic protein synthesis during sepsis in four groups of rats: control, control + TNFbp, septic, and septic + TNFbp. Saline (1.0 ml) or TNFbp (1 mg/kg, 1.0 ml) was injected daily starting 4 h prior to the induction of sepsis. The effect of sepsis and TNFbp administration on hepatic protein synthesis in vivo was examined 5 days later. RESULTS: Sepsis increased the rate of protein synthesis by 35% relative to controls. Accelerated rates of protein synthesis were accompanied by increased total RNA content, eukaryotic initiation factor (eIF) 2alpha content, and phosphorylation of p70S6 kinase. Injection of TNFbp into septic rats for 5 days did not diminish the sepsis-induced stimulation of hepatic protein synthesis. Compared with controls, septic rats treated with TNFbp also showed elevated total RNA content, elF2alpha content, and phosphorylation of p70S6 kinase. No significant differences in any of the parameters measured were observed between untreated and TNFbp-treated septic rats. Treatment of control animals with TNFbp for 5 days was without effect on any of the parameters examined. CONCLUSIONS: TNFbp did not prevent the sepsis-induced stimulation of hepatic protein metabolism or modulate the septic-induced changes in factors regulating protein synthesis. Global rates of protein synthesis in livers from septic rats are accelerated by increases in the abundance or activity of components of translational apparatus.

Animals↗
PubMed2005

Two unique US isolates of Pepino mosaic virus from a limited source of pooled tomato tissue are distinct from a third (European-like) US isolate.

Three strains of Pepino mosaic virus (PepMV) found in the US have been cloned and sequenced by RT-PCR using total RNA from infected tissue as template, and degenerate potexvirus- and PepMV species- and isolate-specific primers. Despite limited source material, the complete nucleotide sequences (6413 and 6410 nts, respectively) of two isolates, PepMV-US1 and PepMV-US2, were obtained and analyzed using total RNA from less than 0.2 g of a pooled infected tomato leaf sample from Arizona. Sequence of the 3'-end of the third isolate from infected fresh tomato fruits from Maryland (PepMV-US3) was also determined. The genome organizations of PepMV-US1 and US2 were typical of the genus Potexvirus, with the following reading frame order: ORF 1, encoding a putative replicase; ORFs 2-4, triple gene block proteins (TGBp) 1-3; and ORF 5, coat protein (CP). Gene-for-gene comparison between PepMV-US1 and US2 revealed the following amino acid identities: 91% in replicase, 89% in TGBp1, 92% in TGBp2, 85% in TGBp3, and 93% in the CP; with an overall nucleotide identity of 86%. Nucleotide sequence comparisons between US1 and US2 and the European isolates showed only 79-82% identity, whereas the identity among the European isolates was over 99%. Sequence comparisons and phylogenetic analysis indicate that PepMV-US1 and US2 are distinctly different from the European isolates, while the CP of PepMV-US3 is nearly identical to the European isolates. The results presented also suggest that TGBp1 and TGBp3 are more suitable than either the replicase or coat protein gene products for discriminating PepMV isolates.

Amino Acid Sequence↗