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Results for “Overlapping PCR”

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At least 631 records · Page 35Linked to original sources

PubMed2006

Ca2+ source-dependent transcription of CRE-containing genes in vascular smooth muscle.

Altered Ca2+ handling has immediate physiological and long-term genomic effects on vascular smooth muscle function. Previously we showed that Ca2+ entry through voltage-dependent Ca2+ channels (VDCCs) or store-operated Ca2+ channels (SOCCs) results in phosphorylation of the Ca2+/cAMP response element (CRE)-binding protein in cerebral arteries. Here, oligonucleotide array analysis was used to determine gene transcription profiles resulting from these two Ca2+ entry pathways in human cerebrovascular smooth muscle cell cultures. Results were confirmed and expanded using quantitative RT-PCR, Western blot, and immunofluorescence. A distinct, yet overlapping, set of CRE-regulated genes was induced by VDCC activation using K+ membrane depolarization vs. SOCC activation by thapsigargin (TG). Membrane depolarization selectively induced a sustained increase in early growth response-1 (Egr-1) mRNA and protein, which were inhibited by the VDCC blocker nimodipine and the SOCC inhibitor 2-aminoethoxydiphenylborate (2-APB). TG selectively induced a sustained increase in MAPK phosphatase-1 (MKP-1) mRNA and protein, and these effects were decreased by 2-APB, but not by nimodipine. The physiological agonist ANG II also stimulated expression of Egr-1 and MKP-1. Coadministration of 2-APB prevented expression of Egr-1 and MKP-1, whereas nimodipine blocked only Egr-1 expression. TG and ANG II induced phosphorylation of ERK, which was sensitive to 2-APB and was selectively required for CRE-binding protein phosphorylation. Our findings thus indicate that Ca2+ entry through VDCCs and store-operated Ca2+ entry can differentially regulate CRE-containing genes in vascular smooth muscle and also imply that agonist-induced signals involved in modulation of gene transcription can be controlled by multiple sources of Ca2+.

Animals↗

PubMed2005

Preparation of ChIL-2 and IBDV VP2 fusion protein by baculovirus expression system.

This study aims to produce an effective subunit vaccine against infectious bursal disease virus (IBDV). The genes of chicken interleukin-2 (ChIL-2) and IBDV viral protein 2 (VP2) were amplified and fused by splice overlap extension-polymerase chain reaction (SOE-PCR). The fusion gene was digested by EcoR I/Kpn I and inserted into pBacPAK8 vector, resulting in recombinant transfer plasmid pBacPakVP2-IL2. The recombinant plasmid was transfected into Sf-9 cells accompanied with hybrid nuclear polyhedrosis virus (HyNPV) genome DNA and lipofectin. Plaque-purification indicated that we had got the recombinant Hy-VP2-IL2. Fusion protein VP2-IL2 was expressed effectively both in insect cells and bombyx mori. The expression of fusion protein was confirmed by ELISA, SDS-PAGE and Western blotting assay, respectively. This efficient system allows us to meet the need for inexpensive vaccines required by the poultry industry.

Animals↗

PubMed2006

[Construction of Lactococcus lactis expression vector of recombinant human trefoil factor family 2].

OBJECTIVE: To construct a Lactococcus lactis expression vector of c-myc-tagged human trefoil factor family 2 (hTFF2) fusion gene to prepare for genetic modification of Lactococcus lactis that can secrete bioactive c-myc-hTFF2 protein. METHODS: Based on the amino sequence of hTFF2 and optimal Lactococcus lactis codon usage, the cDNA of hTFF2 was designed and extended at their 5' ends with a sequence encoding c-myc as the molecular tag. According to the restriction sites of pBluescript II sk (+), the SalI and BamHI sites were arranged at the 5' and 3' ends of the fusion gene respectively. The sequence of the fusion gene c-myc-hTFF2 was designed as 14 oligonucleotides that overlapped with each other, and by means of PCR, all the oligonucleotides were spliced to complete the construction of c-myc-hTFF2 fusion gene. The target gene of c-myc-hTFF2 was inserted into pBluescript II sk (+) to construct the cloning vector pBS-hTFF2 of c-myc-hTFF2 followed by verification by enzyme digestion and DNA sequencing. By digestion of pBS-TFF2 with BamHI/SalI and of pNBC1000 with BamHI/XhoI, we connected c-myc-hTFF2 with pNBC1000 to construct the expression vector c-myc-hTFF2 in E. coli named as pNTFF2. After digestion of pNTFF2 and pTRKH2 with XbaI, the target gene was subcloned into pTRKH2 and the construction of the expression vector pTRTFF2 in Lactococcus lactis was completed. The constructed vector was identified by restriction enzyme digestion. RESULTS AND CONCLUSION: The expression vector pTRTFF2 of c-myc-hTFF2 fusion gene has been successfully constructed. Assembly of oligonucleotides in vitro is an effective means to synthesize the target fusion gene and this prepares the ground for constructing engineered bacterium of Lactococcus lactis.

Cloning, Molecular↗

PubMed1996

Nonallelism of erd and prcd and exclusion of the canine RDS/peripherin gene as a candidate for both retinal degeneration loci.

PURPOSE: To determine whether early retinal degeneration (erd) and progressive rod cone degeneration (prcd), two canine hereditary retinal degenerations, are caused by allelic mutations; to determine the cDNA sequence of the canine RDS/peripherin homolog (CFRDSP); and to test whether mutations(s) in CFRDSP cause(s) either erd or prcd. METHODS: Three erd-affected dogs were crossbred to three prcd-affected dogs, and their progeny were tested by electroretinography and retinal morphology for evidence of retinal degeneration. Canine RDS/peripherin cDNA was cloned and sequenced after reverse-transcription-polymerase chain reaction (RT-PCR) of total retinal RNA. A set of overlapping fragments of CFRDSP cDNA amplified from normal and prcd-affected retinal RNA was examined by double-stranded conformational polymorphism analysis for evidence of any mutation in prcd-affected dogs. RDS/peripherin-specific restriction fragment length polymorphism (RFLP) allelic differences within informative prcd and erd pedigrees were sought by digestion of amplified regions of the CFRDSP gene with different restriction enzymes. A Hinf I RFLP was identified with alleles segregating in a set of prcd and erd informative pedigrees. Linkage of CFRDSP to either prcd or erd was tested using the LINKAGE analysis package. RESULTS: All progeny from the erd x prcd cross were phenotypically normal at ages beyond the age of diagnosis for both parental disorders. The sequence of CFRDSP cDNA is reported (Genbank accession U27349). It is, overall, 79% identical at the nucleotide level with the corresponding human sequence. The coding region shares 89% and 93% nucleotide identity with the corresponding human and feline sequences, respectively. No mutation has been identified in the coding region of CFRDSP in prcd-affected dogs. In prcd pedigrees informative for both prcd and the CFRDSP Hinf I RFLP, a minimum of six obligate recombinants were identified. Similarly, in erd pedigrees, 14 of 29 progeny informative for both erd and this RFLP were obligate recombinants. CONCLUSIONS: The canine erd and prcd mutations are nonallelic. The canine RDS/peripherin gene (CFRDSP) has been excluded as a candidate for both prcd and erd. The demonstrated informativeness of the canine pedigrees on which these studies were based will enable testing other candidate genes for prcd and erd. Sequence information of CFRDSP will enable testing this locus as a candidate in other canine hereditary retinal degenerations.

Alleles↗

PubMed1998

cDNA cloning reveals two mouse beta5 integrin transcripts distinct in cytoplasmic domains as a result of alternative splicing.

The integrin beta5 subunit has only been found to form a heterodimer with subunit alphav which acts as a vitronectin receptor. Integrin alphavbeta5 has been implicated in cell migration and growth factor-induced angiogenesis. In the present study, a mouse liver cDNA library was screened using a human beta5 cDNA fragment obtained by reverse transcriptase PCR (RT-PCR). Three of the clones (MB5, MB15 and MB17) overlapped to give an open reading frame, called beta5A, which is homologous to the human beta5 subunit. The sequence of another clone (MB26), called beta5B, was identical with beta5A, except for a deletion of 29 bp near the 3' end of the open reading frame. The 29 bp deletion resulted in an open-reading-frame shift and a completely different C-terminal sequence in beta5B. beta5A and beta5B were shown, by RT-PCR, to be co-expressed in most mouse tissues tested, although beta5B mRNA was detected at much lower levels than beta5A. beta5A and beta5B mRNAs were also detected in the mouse monocytic cell line, J774, and in isolated mouse peritoneal macrophages. Adhesion of peritoneal macrophages has been shown to up-regulate the expression of both beta5A and beta5B mRNAs. The 29 bp sequence begins with a putative intron-splicing donor site (GTGAT...). A 3' fragment of the mouse integrin beta5 gene was cloned by PCR and sequenced showing that the 29 bp sequence was also immediately followed by an intron. Therefore, the 29 bp sequence was apparently expressed as part of the beta5A mRNA but was spliced out as part of the downstream intron in beta5B. Since the cytoplasmic domains of the integrin beta subunits are important in cytoskeleton attachment and signalling, the two alternatively spliced beta5 isoforms may have distinct roles in cell adhesion and other cellular functions.

Alternative Splicing↗

PubMed2001

The Diversity of Archaea and Bacteria in Association with the Roots of Zea mays L.

The diversity of bacteria and archaea associating on the surface and interior of maize roots (Zea mays L.) was investigated. A bacterial 16S rDNA primer was designed to amplify bacterial sequences directly from maize roots by PCR to the exclusion of eukaryotic and chloroplast DNA. The mitochondrial sequence from maize was easily separated from the PCR-amplified bacterial sequences by size fractionation. The culturable component of the bacterial community was also assessed, reflecting a community composition different from that of the clone library. The phylogenetic overlap between organisms obtained by cultivation and those identified by direct PCR amplification of 16S rDNA was 48%. Only 4 bacterial divisions were found in the culture collection, which represented 27 phylotypes, whereas 6 divisions were identified in the clonal analysis, comprising 74 phylotypes, including a member of the OP10 candidate division originally described as a novel division level lineage in a Yellowstone hot spring. The predominant group in the culture collection was the actinobacteria and within the clone library, the a-proteobacteria predominated. The population of maize-associated proteobacteria resembled the proteobacterial population of a typical soil community within which resided a subset of specific plant-associated bacteria, such as Rhizobium- and Herbaspirillum-related phylotypes. The representation of phylotypes within other divisions (OP10 and Acidobacterium) suggests that maize roots support a distinct bacterial community. The diversity within the archaeal domain was low. Of the 50 clones screened, 6 unique sequence types were identified, and 5 of these were highly related to each other (sharing 98% sequence identity). The archaeal sequences clustered with good bootstrap support near Marine group I (crenarchaea) and with Marine group II (euryarchaea) uncultured archaea. The results suggest that maize supports a diverse root-associated microbial community composed of species that for the first time have been described as inhabitants of a plant-root environment.

Journal Article↗

PubMed1992

Structure of the gene encoding potato cytosolic pyruvate kinase.

The polymerase chain reaction (PCR) has been used to generate a series of overlapping genomic clones representing 43 bp of 5' untranslated sequence, 63 bp of 3' untranslated sequence and the entire coding sequence of the gene encoding potato cytosolic pyruvate kinase (PKc). This portion of the gene is approximately 4.5 kb in length and is interrupted by three introns, one of which is present in the 5' untranslated region. Southern blot analysis indicates that PKc is encoded by a small gene family, and sequence data from a number of PCR-derived genomic clones indicate that there are as many as six PKc genes. Sequence differences between the PCR-generated genomic clones and a PKc cDNA clone are discussed with respect to the fidelity of Taq polymerase. An alignment of intron placement in the potato PKc gene with intron placement in PK genes from other sources indicates that two of the potato introns correspond to intron positions in other species.

Amino Acid Sequence↗

PubMed1989

Automated DNA sequencing methods involving polymerase chain reaction.

Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications. An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers. Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer. DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product. With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99%. An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy. Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities. Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology. Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype.

Autoanalysis↗

PubMed1992

A random STS strategy for construction of YAC contigs spanning defined chromosomal regions.

Sequence tagged sites (STSs) that were generated via Alu-element-mediated polymerase chain reaction (Alu-PCR) and mapped to human Xq26 were used to isolate and overlap yeast artificial chromosomes (YACs). By collating the results of primary pool screening, the order of STSs and YACs was postulated directly. Subsequent isolation of 11 key YACs from 75 positive pools confirmed the proposed contig. Although only a small subset of the available Alu-PCR fragments was used, the STSs were generated at sufficient density to isolate all the YACs required and to identify all except one overlap directly. The results confirmed physical linkage of HPRT to DXS86 and DXS144E. Long-range continuity was determined purely by analysis of the 11 YAC colonies and required no end-rescue. This strategy is therefore an effective approach for the construction of YAC contigs spanning discrete chromosomal regions contained within somatic cell hybrids, with minimal prior knowledge of the region.

Base Sequence↗

PubMed2004

PCR-based approach for sequencing mitochondrial genomes of decapod crustaceans, with a practical example from kuruma prawn (Marsupenaeus japonicus).

An approach for sequencing the entire mitochondrial genomes (mitogenomes) of decapod crustaceans using 79 newly designed and 7 published polymerase chain reaction (PCR) primers is described. The approach comprises the following steps: (1) the entire mitogenome is amplified in 2 or 3 long PCRs; (2) the 86 primers are used in different combinations to amplify contiguous, overlapping short segments of the entire mitogenome with the diluted long PCR products as templates; (3) direct cycle sequencing is conducted using the short PCR products. This strategy allows a more rapid determination of decapod mitogenomic sequences than a traditional method using cloned mitochondrial DNA and primer walking strategy. As a practical example, the mitogenomic sequence for a kuruma prawn Marsupenaeus japonicus (Crustacea: Decapoda), was determined using the PCR-based approach.

Animals↗

PubMed1993

Binding of neutralizing monoclonal antibodies to regions of the fusion protein of respiratory syncytial virus expressed in Escherichia coli.

cDNA containing the entire coding sequence of the respiratory syncytial (RS) virus fusion (F) protein gene (574 amino acids) and two large PstI restriction fragments, encoding amino acids 18 to 212 and 214 to 574, were expressed in Escherichia coli as C-terminal chimeras with beta-galactosidase (beta-gal) in the pEX expression vector system. A further cDNA fragment, overlapping the PstI restriction site and encoding amino acids 190 to 289, was derived by PCR and expressed in a similar manner. Polyclonal rabbit serum raised against RS virus bound to all four chimeric proteins but most strongly to those containing C-terminal sequences. Two monoclonal antibodies (MAbs), 1E3 and RS348, capable of neutralizing the virus and inhibiting the viral fusion function, bound to all chimeras except that derived from the N-terminal PstI fragment, suggesting that their binding sites were located between amino acids 214 and 289. Further analysis of binding to expressed fragments from restriction enzyme digests and PCR amplification demonstrated that both antibodies bound to amino acids 253 to 289. MAb RS348 bound to 12-mer overlapping synthetic peptides containing the sequence 265 to 272 (PITNDQKK) but MAb 1E3 failed to bind to any 12-mer peptide derived from the F protein sequence. Immunization of mice with chimeric proteins containing the whole F protein coding sequence or amino acids 253 to 384, which includes the binding site of the two MAbs identified here, failed to induce antibodies that recognized the native RS virus F protein or could neutralize the virus. This suggests that either the beta-gal partner inhibits the immune response to the protein or that elements missing from the protein expressed in E. coli, perhaps conformational or added post-translation, contribute to the neutralizing antibody epitope.

Amino Acid Sequence↗

PubMed2004

Multiple gene detection by in situ RT-PCR in isolated plant cells and tissues.

With the number of functional genomic approaches in plant biology increasing daily, the demand for rapid and reliable RNA localization techniques for gene characterization is being felt. We present herein a novel, liquid phase in situ RT-PCR (IS-RT-PCR) protocol using a combination of gene-specific fluorescent primers and spectral confocal microscopy to localize target RNA in epicotyl sections and xylogenic suspension cultures of Zinnia elegans. Potential sources of artefacts from fixation to gene detection were systematically eliminated using both fluorescent primers and nucleotides for 18S rRNA gene detection, resulting in a set of optimal parameters for IS-RT-PCR that may be readily adapted to any target gene. By judiciously choosing fluorescent primers with non-overlapping fluorochromes, we have shown that our technique is readily adapted to multiplex IS-RT-PCR, enabling the simultaneous localization of more than one gene within a complex tissue or heterogeneous cell population. A 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein (6-HEX)-labelled primer and a tetrachloro-6-carboxy-fluorescein (TET)-labelled primer were designed for two marker genes associated with programmed cell death in tracheary elements (TEs): an endonuclease (Zen1) and a cysteine protease (ZcP4), respectively. An additional Cyan5 (Cy5)-labelled primer was used to monitor 18SrRNA expression. As expected, the 18S signal was constitutively expressed throughout epicotyls sections and living cells in xylogenic in vitro cultures, whereas Zen1 and ZcP4 were co-localized in forming TEs both in planta and in vitro. Analogous to clustering analysis of gene expression using microarrays to elucidate common metabolic pathways and developmental processes, this novel technique is perfectly adapted to gaining a better understanding of gene function via the coordinated expression of genes in specific cell types of complex tissues and cell populations.

Artifacts↗

PubMed1997

Expression of an F1/V fusion protein in attenuated Salmonella typhimurium and protection of mice against plague.

A novel approach to making fusions of F1 and V antigens, which may be incorporated into a live recombinant vaccine for plague, was developed. The nucleotide sequences encoding Yersinia pestis V antigen (lcrV) and the mature form of F1 antigen (caf1) were amplified by PCR with primers which included tails. At the 3' end of caf1 and the 5' end of lcrV, the tails encoded one of three six- or eight-amino acid linkers or their complementary sequences. The DNA overlap in each linker region was used to prime a second PCR to generate three F1/V fusions, which were cloned into pUC18. The resulting plasmids expressed fusion proteins consisting of F1 and V antigens, separated by the linkers Gly-Ser-Ile-Glu-Gly-Arg, Ser-Ala-Pro-Gly-Thr-Pro or Ser-Ala-Pro-Gly-Thr-Pro-Ser-Arg. As shown by Western blotting of bacterial cell lysates with anti-V and anti-F1 sera, the level of expression and degree of degradation of the three fusion proteins was similar. To investigate the immunogenicity of F1/V, one of the plasmids, placFV6 which encoded the Gly-Ser-Ile-Glu-Gly-Arg linker, was electroporated into the attenuated Salmonella typhimurium strain SL3261 (aroA). Mice receiving two intravenous doses of 5 x 10(6) cfu SL3261/placFV6 developed serum anti-V and anti-F1 IgG titres, with similar IgG1:IgG2a isotype ratios, and T cell responses specific for V and F1 antigens. Six weeks after vaccination, mice were challenged subcutaneously with 7.4 x 10(2) or 7.4 x 10(4) LD50s of Y. pestis strain GB, and a significant degree of protection was demonstrated. These results demonstrate the potential of co-expressing Y. pestis antigens as fusion proteins to develop a live recombinant vaccine against plague.

Amino Acid Sequence↗

PubMed2001

Analysis of enantiomers giving partially overlapped peaks by using different treatments of the chromatographic ultraviolet signals: quantification of pseudoephedrine enantiomers.

Different strategies for the quantification of partially coeluting optical isomers have been investigated. The methods tested are based on the use of different features as the analytical UV signals: peak heights, perpendicular drop areas, first and second derivatives of the chromatograms, peak areas obtained by deconvolution of the overlapped peaks with data fitting optimization, and a multivariate model (principal component regression, PCR). The amphetamine-derivative drug pseudoephedrine was selected as a model compound. For chromatography, LiChrospher 100 RP18 and a mobile-phase consisting of methanol and a solution of carboxymethyl-beta-cyclodextrin (the chiral selector) were used. The UV detector was set at 215 nm. The accuracy obtained with the tested methods at different degrees of overlapping and at different concentration ratios between enantiomers was evaluated. The results of this study demonstrated that the best option for quantification of partially overlapped UV peaks of enantiomers and to obtain the enatiomeric excess is the use of a PCR model using peak heights, perpendicular drop peak areas and deconvoluted peak areas as the original variables. The predictive ability of the proposed calibration model is of about 2-8 times better (depending on the overlapping degree) than that achieved with the other models tested.

Ephedrine↗

PubMed2006

Characterization of the genes encoding UL24, TK and gH proteins from duck enteritis virus (DEV): a proof for the classification of DEV.

Duck enteritis virus (DEV) is classified to the family Herpesviridae, but has not been grouped into any genus so far. Four overlapped fragments were amplified from the DEV genome with polymerase chain reaction (PCR). The assembled length of the four fragments was 6,202 bp, which contained the genes encoding unique long (UL) 24, thymidine kinase (TK) and glycoprotein H (gH) proteins. The UL24 overlapped with TK by 64 nucleotides (nt), in a head-to-head transcription orientation, and the TK and gH had the same transcription orientation. The comparison of amino acid sequences of these 3 deduced DEV proteins with other 12 alphaherpesviruses displayed 5 highly conserved sites in the UL24, as well as another 5 consensus regions in the TK and 4 consensus regions in the gH. The RNA polymerase II transcriptional control elements were identified in all the UL24, TK and gH of DEV. These elements included core promoters, TATA motifs and polyadenylation sites. Phylogenetic analysis for the genetic classification of DEV in the Alphaherpesvirinae subfamily with other 12 alphaherpesviruses was computed. The result showed that DEV was more closely related to avian herpesviruses, except infectious laryngotracheitis virus (ILTV), than to other alphaherpesviruses. Conclusively, according to the phylogenesis-based analysis and the homology comparison of functional domains of UL24, TK and gH, DEV should be classified to a separate genus of the Alphaherpesvirinae subfamily in the family Herpesviridae.

Alphaherpesvirinae↗

PubMed2006

Simplified gene synthesis: a one-step approach to PCR-based gene construction.

PCR-based gene synthesis conventionally requires two steps: first, all overlapping oligonucleotides are assembled by self-priming; then an additional pair of primers is used to amplify the full-length gene product. Here we propose a simplified method of gene synthesis which combines these two steps into one. We have found that the efficiency of this one-step method, which we term "Simplified Gene Synthesis", is affected by multiple parameters of the PCR reactions. In particular, the choice of polymerase is critical for successful one-step assembly. Other important factors include the concentration of assembly oligonucleotides and amplification primers. Moreover, we offer a general method to estimate, given a known mutation rate, how many clones should be sequenced in order to be confident of obtaining at least one correct gene product. Having determined the accuracy of gene products synthesized under optimal conditions with Simplified Gene Synthesis, we show that our estimation works well. Overall, the simplified gene synthesis provides an easier and more efficient approach to gene synthesis, providing a further step towards the future goal of generalized automation for this process.

Cloning, Molecular↗

PubMed1999

New high resolution typing strategy for HLA-A locus alleles based on dye terminator sequencing of haplotypic group-specific PCR-amplicons of exon 2 and exon 3.

In this study, a new sequencing-based typing strategy for the HLA-A locus is presented which involves group-specific separate amplification of exon 2 and 3 of HLA-A alleles in a first step. Conserved HLA-A locus-specific primers of intron 1 or 3 were combined in 10 primer-mixes with group-specific primers hybridizing to the 5'- or 3'-end of exon 3 or 2 for pre-typing of the HLA-A alleles in 14 allelic groups. Maximally four overlapping short amplicons are produced under identical polymerase chain reaction (PCR) conditions with individual separate amplification of exon 2 and exon 3 of the haplotypic alleles in most heterozygous combinations. Time- and money-saving one-directional Big Dye Terminator cycle sequencing is shown to provide reliable high resolution typing of the HLA-A alleles, even in a few cases of two amplicons in one primer reaction mixture. In comparison, to other sequencing-based typing (SBT) techniques the applied typing strategy minimizes the risk of unequal amplification or of drop-outs of one of the haplotypic alleles and allows unequivocal definition of the cis/ trans linkage of polymorphic positions of the complete exon 2 and exon 3 in most heterozygous cells. This also includes detection of new alleles differing in the polymorphic template generating primer annealing sites as well as in unusual combinations of known exon 2 and 3 sequences. With 10 primer sets working under identical conditions for pre-grouping and separate amplification of the haplotypic alleles our SBT procedure also could be implemented in clinical settings of large-scale stem cell donor histocompatibility testing for fast molecular HLA-A matching.

Alleles↗

PubMed1994

A novel member, PC7, of the mammalian kexin-like protease family: homology to PACE4A, its brain-specific expression and identification of isoforms.

By polymerase chain reaction (PCR) with primers corresponding to the sequences of catalytic domain conserved among the mammalian kexin-like protease family, a cDNA fragment encoding a novel member of the family was obtained from rat pituitary. A cDNA for the novel protease was obtained from three overlapping clones isolated from a rat pituitary cDNA library using the PCR product as a screening probe. The protein, designated as PC7, encoded by this cDNA contained a putative activation site with the sequence RXKR, subtilisin-like catalytic domain and a homo B region which are typical of mammalian kexin-like proteases, and short cysteine-rich region at the carboxyl-terminal end. It exhibited surprising sequence similarity to PACE4A. The PC7 transcript was expressed at high levels in the brain. However it was undetectable in the liver, kidney, heart, and spleen. The presence of PC7 isoforms (PC7A and PC7B) was also shown.

Amino Acid Sequence↗