Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Genome quantification”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 847 records · Page 47Linked to original sources
PubMed2007

Rabbit oviductal glycoprotein 1 gene: genomic organization polymorphism analysis and mRNA expression.

The OVGP1 is an oviductal glycoprotein that has positive effects on fertilization and early embryo development. We have amplified and sequenced the rabbit OVGP1 gene, which spans 13 kb and it is formed by 11 exons and 10 introns. To find polymorphisms, a region encompassing the promoter to intron 1 has been sequenced in 22 rabbits of the H, V, R, and A Spanish lines. We have identified five SNPs and one triallelic microsatellite in the promoter region, and three SNPs and one dinucleotide INDEL in intron 1. The 10 polymorphic sites cosegregate forming two haplotypes. The allelic frequencies of the microsatellite have been analyzed in 98 rabbits belonging to the four lines and the three alleles were found in all the lines. The relative quantification of the OVGP1 mRNA in liver, kidneys, lungs, skeletal muscle, ovary, uterus, and oviduct reveals that the OVGP1 expression in the oviduct is 5,500 higher than in the uterus or ovary, whereas a low level of basal expression is detected in non-reproductive tissues. We have also compared the mRNA expression between samples of oviducts taken from non-mated rabbit and samples of oviducts at different stages of the early pregnancy, but no significant differences were found.

Animals↗
PubMed2026

Culture of infectious human norovirus isolated from live contaminated oysters.

Human noroviruses are a major cause of foodborne outbreaks worldwide. Filter-feeding shellfish, such as oysters, can bioaccumulate these viruses in their digestive tissue when grown in sewage-impacted coastal areas and are often implicated in norovirus foodborne outbreaks. Despite the high sensitivity of current molecular assays, these methods for norovirus detection in shellfish fail to distinguish between infectious and non-infectious particles. Assessing norovirus infectivity in shellfish remains a challenge due to the lack of suitable isolation methods that maintain capsid integrity. In this study, a protocol for isolating infectious norovirus from oyster tissues, based on chloroform-butanol elution and polyethylene glycol concentration (CB-PEG), was optimized for the recovery of human norovirus GI and GII. While CB-PEG method recovered various norovirus GI and GII genotypes, it was less efficient at the genomic level than a protocol based on proteinase K elution (adapted from ISO 15216) and showed genotype-dependent viral recovery rates. By optimizing the flocculation step, we improved the method's compatibility with human intestinal enteroid (HIE) cultures. Using this approach, we successfully quantified infectious norovirus GII.3 titers recovered from artificially-contaminated live oysters. Interestingly, infectious virus was better isolated following a freezing step of the digestive tissues, with titers ranging from 13 to 40 TCID50/mL for positive samples. In conclusion, this study established an optimized methodological approach for the relative quantification of infectious norovirus GII.3 in shellfish, paving the way for future research on viral persistence and inactivation strategies in this foodstuff.

Norovirus↗
PubMed2002

Molecular diagnostics in infectious diseases and public health microbiology: cottage industry to postgenomics.

Molecular methods have been used increasingly over the past ten years to improve the sensitivity and speed of diagnosis in infectious diseases. Although their routine use is still limited to the detection of pathogens that are difficult to culture in vitro,'real-time' methods, commercial kits, quantification and automation will increase potential applications. Molecular methods are now widely used for epidemiological fingerprinting of isolates of public health importance. Sequence-based identification and strain typing, together with the development of tools that can probe for thousands of markers, will allow detailed strain fingerprinting to assist in disease management and control.

Animals↗
PubMed1998

Molecular detection of cancer cells by competitive reverse transcription-polymerase chain reaction analysis of specific CD44 variant RNAs.

BACKGROUND: CD44 is a cell surface glycoprotein implicated in such diverse biologic processes as lymphocyte activation and homing, extracellular matrix adhesion, and cellular migration. Primary transcripts of the CD44 gene can be alternatively spliced to produce a variety of messenger RNA (mRNA) species. The standard form of CD44 mRNA contains sequences from at least 20 genomic exons; variant mRNAs contain sequences from one or more additional exons (v1-10). Predominant expression of a specific CD44 variant, i.e., CD44v8-10, in several human carcinomas has been described previously. In this study, we developed a novel molecular approach for detecting cancer cells that overexpress CD44v8-10 mRNA. METHODS: After finding that CD44v8-10 was predominantly expressed in non-small-cell lung and bladder carcinomas and that CD44v10 was predominantly expressed in leukocytes, we developed a competitive reverse transcription-polymerase chain reaction assay (CC-RT-PCR) that allows quantification of the relative expression of these two mRNA species in clinical specimens (i.e., determination of a v8-10/v10 ratio). CC-RT-PCR analysis was applied to pleural effusion specimens from patients with benign or malignant lung diseases as well as to spontaneously voided urine samples from patients with benign or malignant urologic diseases. RESULTS: Fifty two of 54 samples from patients with benign diseases expressed CD44v10 predominantly (v8-10/v10 ratio < or = 0.65), whereas 46 of 61 samples from patients with malignant diseases expressed CD44v8-10 predominantly (v8-10/v10 ratio > 1.00) (two-sided P < .001). CC-RT-PCR detected predominant expression of CD44v8-10 in cytologically negative samples from 11 patients who were later diagnosed with malignant disease. CONCLUSIONS: CC-RT-PCR analysis of CD44v8-10 expression could be an important adjunct to cytologic examination in cancer diagnosis, especially in detecting exfoliated cancer cells in pleural effusions and urine.

Breast Neoplasms↗
PubMed2004

Detecting imbalanced expression of SNP alleles by minisequencing on microarrays.

BACKGROUND: Each of the human genes or transcriptional units is likely to contain single nucleotide polymorphisms that may give rise to sequence variation between individuals and tissues on the level of RNA. Based on recent studies, differential expression of the two alleles of heterozygous coding single nucleotide polymorphisms (SNPs) may be frequent for human genes. Methods with high accuracy to be used in a high throughput setting are needed for systematic surveys of expressed sequence variation. In this study we evaluated two formats of multiplexed, microarray based minisequencing for quantitative detection of imbalanced expression of SNP alleles. We used a panel of ten SNPs located in five genes known to be expressed in two endothelial cell lines as our model system. RESULTS: The accuracy and sensitivity of quantitative detection of allelic imbalance was assessed for each SNP by constructing regression lines using a dilution series of mixed samples from individuals of different genotype. Accurate quantification of SNP alleles by both assay formats was evidenced for by R2 values > 0.95 for the majority of the regression lines. According to a two sample t-test, we were able to distinguish 1-9% of a minority SNP allele from a homozygous genotype, with larger variation between SNPs than between assay formats. Six of the SNPs, heterozygous in either of the two cell lines, were genotyped in RNA extracted from the endothelial cells. The coefficient of variation between the fluorescent signals from five parallel reactions was similar for cDNA and genomic DNA. The fluorescence signal intensity ratios measured in the cDNA samples were compared to those in genomic DNA to determine the relative expression levels of the two alleles of each SNP. Four of the six SNPs tested displayed a higher than 1.4-fold difference in allelic ratios between cDNA and genomic DNA. The results were verified by allele-specific oligonucleotide hybridisation and minisequencing in a microtiter plate format. CONCLUSIONS: We conclude that microarray based minisequencing is an accurate and accessible tool for multiplexed screening for imbalanced allelic expression in multiple samples and tissues in parallel.

Alleles↗
PubMed2006

Development and validation of a rotor-gene real-time PCR assay for detection, identification, and quantification of Chlamydia trachomatis in a single reaction.

A multitarget real-time PCR (MRT-PCR) for detection of Chlamydia trachomatis DNA was developed and validated. There were three targets for amplification in a single reaction: the cryptic plasmid (CP), the major outer membrane protein (MOMP) gene, and an internal control. The assay had the following characteristics: (i) detection and confirmation of the presence of C. trachomatis DNA in a single reaction, (ii) detection of all genovars of C. trachomatis without any cross-reactivity with pathogenic bacteria or commensal organisms of the oropharynx and genital tract, (iii) a 95% probability of detection with three copies of MOMP and one copy of CP per reaction mixture, (iv) identification of the inhibition of amplification, (v) a quantitative dynamic range of 25 to 250,000 genome copies per reaction mixture, (vi) high intra- and interassay reproducibilities, and (vii) correct identification of all samples in the validation panel. There were 146 COBAS Amplicor PCR (Amplicor PCR)-positive samples and 122 Amplicor PCR-negative samples in the panel. MRT-PCR detected CP DNA alone in 6 (4%) Amplicor PCR-positive samples and both CP and MOMP DNAs in 140 (96%) of 146 Amplicor PCR-positive samples. The quantity of MOMP DNA in 95 (68%) of 140 samples was within the dynamic range of the assay. The median C. trachomatis load in these samples was 321 genome copies per reaction mixture (range, 26 to 40,137 genome copies per reaction mixture). Due to the inclusion of two different C. trachomatis-specific targets, the assay confirmed 259 (97%) of 268 results in a single reaction. This assay could be used in the qualitative format for the routine detection of C. trachomatis and in the quantitative format for study of the pathogenesis of C. trachomatis-associated diseases. The assay demonstrated the potential to eliminate the need for confirmatory testing in almost all samples, thus reducing the turnaround time and the workload.

Bacterial Outer Membrane Proteins↗
PubMed1996

Quantification of hepatitis C virus-infected peripheral blood mononuclear cells by in situ reverse transcriptase-polymerase chain reaction.

Hepatitis C virus (HCV) is known to infect peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis C, but the proportion of HCV-infected circulating cells is not detectable by conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and the pathogenic significance of HCV lymphotropism is still unclear. Therefore, we have devised an in situ RT-PCR technique using fluorescein-labeled HCV-specific primers revealed by flow cytometry. PBMC were isolated from 28 patients with chronic HCV-related liver disease; of these, 6 had previously received an orthotopic liver transplantation (OLT) and were on immuno-suppressive treatment. Fourteen patients (50%) were found positive for HCV genome within PBMC by in situ RT-PCR, the proportion of HCV-infected cells ranging from 0.2% to 8.1%. All 6 OLT patients tested positive. The fluorescent signal, corresponding to the HCV-specific 340-bp amplicon, was confined to part of the cytoplasmic compartment of scattered PBMC. Of these 14 patients, 12 had also negativestrand HCV RNA within PBMC detected by "tagged" RT-PCR. We conclude that HCV may infect a significant proportion of PBMC in chronic hepatitis C patients, especially immunosuppressed OLT cases, and that viral replication within PBMC is a common occurrence. Over time, the persistence of HCV-infected immune system cells might interfere with normal immunologic mechanisms and play a role in the pathogenic processes leading to extrahepatic disorders such as mixed cryoglobulinemia and B-cell malignant lymphoma.

Adult↗
PubMed2005

Genetic analysis of dry matter and nitrogen accumulation and protein composition in wheat kernels.

The maximum rate and duration for grain dry matter (DM) and nitrogen (N) accumulation were evaluated in 194 recombinant inbred lines (RILs) from a cross between the two French wheat cultivars Récital and Renan. These cultivars were previously identified as having contrasting kinetics of grain DM and N accumulation. Grain protein composition was analysed by capillary electrophoresis (CE), which enabled quantification of the different storage protein fractions (alphabetagamma-gliadins, omega-gliadins, LMW glutenins, HMW glutenins, and each of their subunits). Correlation analyses revealed that DM and N accumulation rates were closely correlated and repeatable over several years, which was not the case for DM and N accumulation durations, and that protein composition was primarily influenced by the N accumulation rate. This was particularly true for the LMW-glutenins and the alphabetagamma-gliadins, the most abundant protein fractions. A genetic map of 254 molecular markers covering nearly 80% of the wheat genome was used for quantitative trait loci (QTL) analysis. A total of seven QTLs were found. Five QTLs were significantly associated with the kinetics of DM and N accumulation, and two of them also influenced protein composition. Two QTLs affected only the protein composition. One major QTL explained more than 70% of the total variation in HMW-GS Glu1B-x content.

Chromosome Mapping↗
PubMed2006

Assessment of biases associated with profiling simple, model communities using terminal-restriction fragment length polymorphism-based analyses.

Community profiles based on terminal-restriction fragment length polymorphism (T-RFLP) analyses of amplified ribosomal RNA genes are used to monitor changes in microbial community structure and are sometimes employed for semi-quantitative estimates of species richness and abundance in environmental samples. To assess the accuracy of T-RFLP community profiles representing the relative abundance of bacteria in a sample, five species of ruminal bacteria were used to construct simple "communities". Template DNA for PCR amplification was generated either by mixing equal quantities of genomic DNA from pure cultures or by mixing equal numbers of cells prior to DNA extraction. Pairwise mixtures of Fibrobacter succinogenes S85 with Ruminococcus albus 8, Ruminococcus flavefaciens FD-1, Butyrivibrio fibrisolvens 49 and Streptococcus bovis JB1 were created and a 5-member community was constructed. With genomic DNA mixes, relative abundance calculations based on T-RFLP patterns did not reflect input ratios. These discrepancies could not be accounted for by differences in genome size and rRNA operon copy number. In cell mixing experiments, easily lysed cells were overrepresented. To determine if a numerical correction factor could be used to compensate for observed discrepancies, we attempted to quantify biases attributed to DNA extraction and PCR amplification. Biases attributable to these factors led to deviations from expected PCR product ratios by 6% to 38%. We found that interactions were so complex that a suitable factor could not be derived. The unsystematic dependence of T-RFLP peak ratios on variability of DNA extraction and PCR amplification prevents accurate quantification of the relative abundance of microorganisms designed to represent simplified natural populations.

Bacteria↗
PubMed1985

Biochemical studies of rat atrial natriuretic factor.

The natriuretic substances were purified from rat atrium (ANF, atrial natriuretic factor) and were shown to be identical with the inhibitor of norepinephrine-induced contraction of smooth muscle. Their four native forms were isolated. Amino acid sequence analyses showed they are peptides with 35, 31, 30 and 25 amino acid residues respectively and contain a ring structure consisting of 17 amino acid residues and a disulfide bridge. The presence of a high molecular weight prohormone was shown. cDNA coding for the precursor was cloned and used to deduce the amino acid sequence of the preprohormone. Genomic DNA for ANF was cloned and the presence of two introns were found. Several ANF peptides were synthesized. Structure-function studies showed that the ring structure is essential for the activity. Antibodies produced against the synthetic 25 amino acid residue ANF were used to develop radioimmunoassay. The presence of ANF in rat plasma was demonstrated as evidence that ANF is a circulating hormone. ANF was also found in the hypothalamus of rats. The quantitative determination of the synthetic ability of ANF has been determined by the application of ANF cDNA for the quantification of ANF messenger RNA. Immunohistochemical methods localized ANF in cardiac atriocytes, gonadotrophs in anterior pituitary and adrenal medulla (chromaffin cells). A strong immuno-reactivity was found in dark cells of the collecting ducts of the kidney. ANF increases cyclic GMP in target cells suggesting that cyclic GMP may be the intracellular mediator of ANF action.

Adrenal Cortex↗
PubMed2005

[Evaluation of the agreement of results obtained from ABI Prism 7000 and 7700 sequencers in the quantification of hepatitis B virus DNA].

There are different methods and systems for quantification of HBV-DNA in clinical virology laboratories. The aim of this study was to evaluate the agreement of the polymerase chain reaction (PCR) protocol with ABI Prism 7000 instrument (PE Biosystems) which was designed and optimised for ABI Prism 7700 (PE Biosystems). Serum samples obtained from 168 chronic hepatitis B patients were treated with "High Pure Viral Nucleic Acid Kit" (Roche Applied Science, USA), and MagnaPure LC isolation station (Roche Applied Science, Germany) was used for HBV-DNA isolation. Real time PCR procedure which amplifies pre-S gene of HBV genome was performed. Amplification and detection steps of all samples were performed with ABI Prism 7700 and 7000 Sequence Detection Systems. Among 168 samples, results of 124 serum samples were found to be in dynamic ranges of the tests. The results of these 124 samples obtained from ABI 7000 and ABI 7700 were concordant. Among the rest of 44 samples; one yielded higher than 10(10) copies/mL with two of the systems; six samples gave results higher than 10(10) copies/mL only with 7700; thirty samples were found negative with both of the systems; seven samples were positive (320-1220 copies/mL) with 7000 but negative with 7700. As a result this PCR protocol can be used in ABI 7000 system according to viral quality control (VQC) results. However, since the results of samples with HBV-DNA less than 1 x 10(6) copy/ml were discordant with the results obtained by ABI 7700 system, it can be concluded that different systems must not be used for the management and monitoring of the same patient.

DNA, Viral↗
PubMed1990

Chromosome-specific identification and quantification of S1 nuclease-sensitive sites in yeast chromatin by pulsed-field gel electrophoresis.

Sites that are sensitive to the single-strand-specific endonuclease S1 ('S1-sensitive sites', SSS) occur in native chromatin and, like DNA double-stranded breaks (DSB), they are induced by DNA-damaging agents, such as ionizing radiation. We have developed a method to quantify SSS and DSB in yeast chromatin by using pulsed-field gel electrophoresis (PFGE) to separate the intact chromosomal-length DNA molecules from the lower molecular-weight broken ones. Direct evaluation of the photonegatives of the ethidium bromide-stained gels by laser densitometry enabled us to calculate the numbers of DSB and SSS per DNA molecule. These numbers were determined from the bulk of the non-separated genomic DNA of yeast, corresponding to a single band in the PFGE (pulse time 10 seconds), and in each of the eight largest yeast chromosomes, corresponding to distinct bands in the PFGE gels (pulse time 50 seconds), which were not superimposed by the smear of the broken, low molecular-weight DNA. Furthermore, the induction of DSB and SSS in a specific chromosome (circular chromosome III) was determined by Southern hybridization of the PFGE gels with a suitable centromere probe, followed by densitometry of the autoradiographs. Our method allows the chromosome-specific monitoring of DSB and all those DNA structures that are processed either in vivo or in vitro into DSB and which may not be distributed randomly within the genome.

Chromatin↗
PubMed2006

Performance of the automated Abbott RealTime HIV-1 assay on a genetically diverse panel of specimens from Brazil.

The combination of automated sample preparation and real-time RT-PCR for measurement of HIV-1 viral load has the potential to significantly enhance throughput, reduce operator-associated error, and increase assay sensitivity and dynamic range. In this study, RNA was extracted from the plasma of 91 HIV-1 seropositive Brazilian blood donors using the Abbott m2000sp automated sample preparation system. Viral loads measured using the RealTime HIV-1 (RealTime HIV-1) assay and the Abbott m2000rt instrument were compared to values obtained in the LCx HIV RNA quantitative assay. Subtype was determined for 89 of 91 specimens by sequence/phylogenetic analysis of three genomic regions: gag p24, pol integrase, and env gp41. The panel included 69 subtype B, 1 C, 2 F, and 17 recombinant strains. Eighty-seven specimens were quantified by both assays. Two specimens were quantified only in RealTime HIV-1. Two additional specimens below the detection limit of both assays were also negative on PCR amplification. Viral load results were highly correlated, and good agreement was observed between assays with 90% of values within 0.5 log(10)copies/ml. The RealTime HIV-1 assay and m2000 system offer the advantages of automation while providing reliable quantification of diverse HIV strains.

Automation↗
PubMed2006

Relationship between Marek's disease virus load in peripheral blood lymphocytes at various stages of infection and clinical Marek's disease in broiler chickens.

Vaccination with herpesvirus of turkey (HVT) vaccine provides protection against clinical Marek's disease (MD) but does not preclude infection with wild-type MD virus (MDV). The quantity of MDV detected in circulating lymphocytes during the early period after infection may be a useful predictor of subsequent clinical MD later in the life. A study was designed to quantify MDV and HVT copy number in peripheral blood lymphocytes (PBL) using real-time polymerase chain reaction between days 5 and 35 post-challenge and to relate this to subsequent development of gross MD lesions. Female commercial broiler chickens were vaccinated with HVT or were sham-vaccinated at hatch, then challenged with MDV strain MPF-57 at day 2 post-vaccination and reared in positive-pressure isolators up to 56 days post-challenge, when all survivors were euthanized. All dead and euthanized chickens were examined post mortem for gross MD lesions. Birds were scored for MD lesions and mortality. MDV and HVT genome copy numbers were determined for each PBL sample. There was an increase in HVT load in PBL between days 7 and 37 post-vaccination, with marked increases between days 7 and 16 and again between days 30 and 37. There was a steady increase in MDV load to 35 days post-challenge. The mean MDV copy number (log(10)) was greater in chickens subsequently exhibiting gross MD lesions (5.05 +/- 0.21) than in those that did not (2.88 +/- 0.223), with the largest difference at 14 and 21 days post-challenge (P < 0.001). Quantification of MDV during early infection is therefore a potential tool for monitoring MD in broiler flocks.

Animals↗
PubMed2004

Isolation of genes associated with developmentally competent bovine oocytes and quantitation of their levels during development.

We have performed suppressive subtraction hybridization (SSH) of populations of developmentally competent and incompetent bovine oocytes from large (> or =5-mm) and small (< or =2-mm) follicles to isolate messenger RNA associated with the attainment of developmental competency. RNA was amplified in a linear fashion and then subjected to the SSH procedure to produce a library enriched for genes associated with competency. One thousand clones of this library were subjected to a differential screening approach to identify 31 potentially upregulated isolates. Sequencing revealed these to represent 21 genes. To rigorously identify the degree of upregulation and reproducibility thereof, we examined the expression of these genes in three separate pools of developmentally competent and incompetent oocytes by quantitative real-time PCR. Results indicated that upregulation varied from zero to threefold, showing that accurate quantification is essential for the interpretation of such differential screening experiments. Furthermore, it appears that the molecular causes for poor developmental capacity may be highly complex and be reliant on many small changes. We further characterized a selection of these novel and known maternally expressed genes for their absolute expression levels during maturation in the presence or absence of an inhibitor of transcription and during preattachment development. Last, the effect of nuclear transfer on the levels of these genes was assayed. Nuclear transfer was found to differentially affect transcript levels of genes expressed after embryonic genome activation but did not prevent the degradation of maternal transcripts or result in activation of maternal genes that are silent at blastocyst stages.

Animals↗
PubMedDate not supplied

Biocontrol strain Pseudomonas sp. W34: specific detection and quantification in the rhizosphere of Cucumis sativus with a DNA probe and genotypic characterization by DNA fingerprinting.

A DNA probe specific for biocontrol strains of Pseudomonas was produced by screening randomly amplified polymorphic DNA (RAPD) PCR fragments. Specificity of the probe was assessed by dot blot and colony hybridization. It was used to specifically determine the population of these strains on roots of Cucumis sativus cv. Delikatess. Two polymorphic RAPD fragments of 750 bp, and 550 bp showed identical specificity. The biocontrol strain Pseudomonas sp. W34 was shown to be competitive in the rhizosphere of cucumber and to maintain a stable population for at least 10 days when inoculated on the seed. The phylogenetic relationships between the biocontrol and reference strains were analyzed at the strain level by means of RAPD and repetitive sequence-based PCR genomic fingerprinting (rep-PCR), and at higher taxonomic levels by means of amplified 16S ribosomal DNA restriction analysis ARDRA. It was shown that the antagonistic strains are closely related, forming a separate cluster from other non-antagonistic and reference Pseudomonas strains, their taxonomic placement remaining uncertain.

Cucumis sativus↗
PubMed2006

Quantitative analysis of messenger RNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, beta-actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitro.

Real-time reverse transcription PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, measurement of gene expression generally requires selection of a valid reference (housekeeping gene) for data normalization to compensate for inherent variations. Given the dynamic nature of early embryonic development, application of this technology to studies of oocyte and early embryonic development is further complicated due to limited amounts of starting material and a paucity of information on constitutively expressed genes for data normalization. We have validated quantitative procedures for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA abundance during bovine meiotic maturation and early embryogenesis and utilized this technology to determine temporal changes in mRNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, and histone H2A. Quantification of amounts of specific exogenous RNAs added to samples revealed acceptable rates of RNA recovery and efficiency of reverse transcription with minimal variation. Progression of bovine oocytes to metaphase II resulted in reduced abundance of polyadenylated, but not total transcripts for majority of above genes; however phosphoglycerokinase exhibited a significant decline in both RNA populations. Abundance of mRNAs for above genes in early embryos generally remained low until the blastocyst stage, but abundance of ribosomal protein L-15 mRNA was increased at the morula stage and histone H2A mRNA showed dynamic changes prior to embryonic genome activation. Results demonstrate a valid approach for quantitative analysis of mRNA abundance in oocytes and embryos, but do not support constitutive expression of above genes during early embryonic development.

Actins↗
PubMed1992

Human serum amyloid A protein. Complete amino acid sequence of a new variant.

Serum amyloid A protein (SAA), an acute-phase reactant and apolipoprotein of high-density lipoprotein, is a polymorphic protein with six reported isoforms. These are the products of three genes, i.e., cDNA pA1, cDNA pSAA82 and genomic DNA SAAg9, the last two being allelic variants at a single locus. We have identified an individual with additional novel SAA isoforms on isoelectric-focusing analysis. By using 3-bromo-3-methyl-2-(2'-nitrophenylsulphenyl)-indolenine (BNPS-skatole) cleavage of the protein at tryptophan residues we obtained the complete amino acid sequence of a novel isoform. Additional cleavage by endoproteinase Asp-N allowed verification of the tryptophan residues and complete amino acid sequence of both isoforms. The suitability of this approach to the rapid sequencing of SAA was demonstrated. Sequence analysis and quantification suggest that these isoforms are the result of the first confirmed allelic variation at the SAA1 locus. We designate the protein products of this allele SAA1 beta (pI 6.1) and SAA1 beta des-Arg (pI 5.6).

Amino Acid Sequence↗