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Results for “Overlapping PCR”

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PubMed2001

Uniform integration of genome mapping data using intersection graphs.

MOTIVATION: The methods for analyzing overlap data are distinct from those for analyzing probe data, making integration of the two forms awkward. Conversion of overlap data to probe-like data elements would facilitate comparison and uniform integration of overlap data and probe data using software developed for analysis of STS data. RESULTS: We show that overlap data can be effectively converted to probe-like data elements by extracting maximal sets of mutually overlapping clones. We call these sets virtual probes, since each set determines a site in the genome corresponding to the region which is common among the clones of the set. Finding the virtual probes is equivalent to finding the maximal cliques of a graph. We modify a known maximal-clique algorithm such that it finds all virtual probes in a large dataset within minutes. We illustrate the algorithm by converting fingerprint and Alu-PCR overlap data to virtual probes. The virtual probes are then analyzed using double-linkage intersection graphs and structure graphs to show that methods designed for STS data are also applicable to overlap data represented as virtual probes. Next we show that virtual probes can produce a uniform integration of different kinds of mapping data, in particular STS probe data and fingerprint and Alu-PCR overlap data. The integrated virtual probes produce longer double-linkage contigs than STS probes alone, and in conjunction with structure graphs they facilitate the identification and elimination of anomalies. Thus, the virtual-probe technique provides: (i) a new way to examine overlap data; (ii) a basis on which to compare overlap data and probe data using the same systems and standards; and (iii) a unique and useful way to uniformly integrate overlap data with probe data.

Algorithms↗
PubMed2006

[Gene's construction, expression and activity analysis of humanized single-domain antibody against human lung cancer].

AIM: To construct the gene of humanized single-domain antibody hu3D3V(H) against human lung cancer, to express it in E.coli and analyze its activity. METHODS: The mAb3D3V(H) was humanized by using CDRs grafting technique. The hu3D3V(H) gene was assembled by overlapping PCR. The expression vector pET22(b+)/hu3D3V(H) was constructed and transformed into E.coli BL21(DE3) and the hu3D3V(H) protein was expressed under IPTG induction. After purification by Ni(2+)-affinity chromatographic column, the activity of hu3D3V(H) protein was analyzed by indirect ELISA and competitive inhibition ELISA. RESULTS: The target gene with correct sequence was obtained by overlapping PCR. The hu3D3V(H) protein was expressed as inclusion bodies with the yield of more than 30% of total bacterial proteins. After purification, the purity of hu3D3V(H) was more than 95%. The reactivity of purified protein was the same as parent antibody, and could inhibit the binding of mAb3D3 to L342 cells. CONCLUSION: The hu3D3V(H) still retains the reactivity and specificity of mAb3D3, which lays the foundation for its further clinical application.

Antibodies↗
PubMed1996

Cloning and characterization of a glycogen synthase cDNA from human endometrium.

One major human uterine response to post-ovulatory progesterone is the accumulation of glycogen by the endometrium. A temporally related increase in glycogen synthase activity has been documented, but the isozyme responsible has not yet been identified. We have amplified a glycogen synthase (GS) complementary DNA (cDNA) from human endometrium by reverse transcription-polymerase chain reaction (RT-PCR). Overlapping clones of the PCR products provided a cDNA that is 3534 base pairs (bp) long, including a 22-bp poly(A)+ tail, and an open reading frame that encodes a 737 amino acid protein with a molecular weight of 83936. This cDNA is almost identical to that of human striated muscle GS. Differences include a double nucleotide substitution at 1983-1984 and five single nucleotide substitutions located, respectively, at positions 379, 2457, 2470, 2477, and 2553. These differences only alter the predicted amino acid sequence from that of the striated muscle protein by a single substitution at position 608. A 5'-end fragment plus an internal fragment of human myometrial GS cDNA were also analysed and were shown to have identity with the endometrial GS cDNA. Northern blot hybridization, using a human muscle-derived cDNA probe, detected the presence of a 4.0-kb GS messenger RNA (mRNA) in the endometrium and myometrium. Our results establish that the GS of human Mullerian tissues is, essentially, identical to that reported for human striated muscle.

Amino Acid Sequence↗
PubMed2002

Structure--function studies of the recombinant protein bioemulsifier AlnA.

Acinetobacter radioresistens KA53 produces an extracellular bioemulsifier, referred to as alasan. The surface active component of alasan is a 35.77 kDa protein AlnA. Although AlnA and the Escherichia coli outer membrane protein A (OmpA) have a high amino acid sequence homology, E. coli OmpA has no emulsifying activity. Comparison of the amino acid sequences of AlnA and E. coli OmpA revealed four hydrophobic regions in AlnA that were absent in E. coli OmpA. Deletions and substitutions (with the homologous OmpA sequence) were constructed in each of the four hydrophobic regions of AlnA by site-directed polymerase chain reaction (PCR) mutagenesis, using the overlap PCR method. Analysis of the emulsifying activities of the mutated AlnA molecules demonstrated that all four hydrophobic regions were necessary for emulsifying activity. However, most of the inactive mutated proteins still adhered avidly to hexadecane. These findings indicate that in addition to binding to hydrocarbons, the protein emulsifier must form a specific structure on the surface of the hydrocarbon that prevents coalescence of oil droplets. This is the first structure-function study of a protein bioemulsifer.

Acinetobacter↗
PubMed1999

Sequence heterogeneity within three different regions of the hepatitis G virus genome.

Two sets of primers derived from the 5'-terminal region and the NS5 region of the hepatitis G virus (HGV) genome were used to amplify PCR fragments from serum specimens obtained from different parts of the world. All PCR fragments from the 5'-terminal region (5'-PCR, n = 56) and from the NS5 region (NS5-PCR, n = 85) were sequenced and compared to corresponding published HGV sequences. The range of nucleotide sequence similarity varied from 74 and 78% to 100% for 5'-PCR and NS5-PCR fragments, respectively. Additionally, five overlapping PCR fragments comprising an approximately 2.0-kb structural region of the HGV genome were sequenced from each of five sera obtained from three United States residents. These sequences were compared to 20 published sequences comprising the same region of the HGV genome. Nucleotide and deduced amino acid sequences obtained from different individuals were homologous from 82.9 to 93. 6% and from 90.4 to 99.0%, respectively. Sequences obtained from follow-up specimens were almost identical. Comparative analysis of deduced amino acid sequences of the HGV structural proteins and hepatitis C virus (HCV) structural proteins combined with an analysis of predicted secondary structures and hydrophobic profiles allowed prediction of processing sites within the HGV structural proteins. A phylogenetic sequence analysis performed on the 2.0-kb structural region supports the existence of three previously identified HGV genetic groups. However, phylogenetic analysis performed on only small DNA fragments yielded inconsistent genetic grouping and failed to confirm the existence of genetic groups. Thus, in contrast to HCV where almost any region can be used for genotyping, only large or carefully selected genome fragments can be used to identify consistent HGV genetic groups.

5' Untranslated Regions↗
PubMed2006

Molecular cloning and characterisation of a thioester-containing alpha2-macroglobulin (alpha2-M) from the haemocytes of mud crab Scylla serrata.

Molecular approaches were used to clone thioester-containing alpha2-macroglobulin (alpha2-M) genes in the haemocytes of mud crab Scylla serrata. The full length sequence of alpha2-M was determined by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the alpha2-M cDNA clone consists of 5491bp with an open reading frame (ORF) of 4986bp encoding a protein of 1662 amino acids with 22 residues signal sequence. The calculated molecular mass of the mature protein is 184.2kDa with an estimated pI of 8.41. The S. serrata alpha2-M sequence contains putative functional domains including a GCGEQNM thioester region, a bait region, and a receptor-binding domain which are present in other invertebrate and vertebrate alpha2-Ms. Sequence comparison showed that alpha2-M deduced amino acid sequence of S. serrata has an overall similarity of 68% and 48% to that of kuruma shrimp Marsupenaeus japonicus and American horseshoe crab Limulus polyphemus, respectively. Phylogentic analysis revealed that S. serrata alpha2-M is closely related to other arthropod alpha2-M, and displays the highest similarity to M. japonicus alpha2-M. The alpha2-M was mainly expressed in haemocytes. Quantitative real-time RT-PCR analysis showed that alpha2-M mRNA transcript in haemocytes of S. serrata increased significantly in 24h- and 48h-post lipopolysaccharide (LPS) injection.

Actins↗
PubMed2006

Molecular cloning and characterisation of a proteinase inhibitor, alpha 2-macroglobulin (alpha2-M) from the haemocytes of tiger shrimp Penaeus monodon.

An alpha 2-macroglobulin (alpha2-M) gene was cloned from the haemocytes of tiger shrimp Penaeus monodon by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the alpha2-M cDNA consists of 4876 bp with an open reading frame (ORF) of 4494 bp, a 52 bp 5'-untranslated region, and a 327 bp 3'-untranslated region containing a poly A signal. The open reading frame encodes a protein of 1498 amino acids with 18 residues signal sequence. The predicted molecular mass of the mature protein (1480 amino acids) is 167.7 kDa with an estimated pI of 5.30. The P. monodon alpha2-M sequence contains putative functional domains including a GCGEQNM thioester region, a bait region, and a receptor-binding domain which are present in other invertebrate and vertebrate alpha2-Ms. Sequence comparison showed that alpha2-M deduced amino acid sequence of P. monodon has an overall similarity of 85, 52 and 49% to that of kuruma shrimp Marsupenaeus japonicus, American horseshoe crab Limulus polyphemus and mud crab Scylla serrata, respectively. Alignment of the deduced amino acid sequence to other species alpha2-M showed that the overall structure is evolutionarily conserved and phylogenetic analysis revealed that P. monodon alpha2-M is closely related to other arthropod alpha2-M, and displays the highest similarity to M. japonicus alpha2-M. The alpha2-M was mainly expressed in haemocytes, but not in eyestalk, gill, muscle, hepatopancreas, and intestine. Quantitative real-time RT-PCR analysis showed that alpha2-M mRNA transcript in haemocytes of P. monodon increased significantly in 12, 24 and 48 h post-peptidoglycan (PG) injection, but returned to the original values in 72 h post-PG injection.

Amino Acid Sequence↗
PubMed2005

[Expression of epidermal growth factor-adenovirus E4orf4 fusion protein in tumor cells and its cytotoxicity].

BACKGROUND & OBJECTIVE: Human epidermal growth factor (EGF), an important growth factor, may stimulate cell growth and proliferation. EGF receptor (EGFR) expresses on the surface of normal cells, and abnormally over-expresses on many kinds of tumor cells, especially on solid tumor cells. Adenovirus early region 4 open reading frame 4 protein (E4orf4) is a novel cytotoxin that can specifically induce p53-independent apoptosis in tumor cells. Based on the targeting of EGF and cytotoxicity of E4orf4, we proposed to design a novel fusion protein at molecular level by recombining EGF and E4orf4 to target and then kill tumor cells. METHODS: EGF and E4orf4 coding sequences were amplified by polymerase chain reaction (PCR), and then genetically fused by overlapping PCR. EGF-E4orf4 fragment was cloned into the yeast expression vector. Recombinant plasmid DNA was transformed into the yeast Pichia pastoris. Fusion proteins were purified by SP Sepharose ion exchange chromatography. Cytotoxicity of EGF-E4orf4 on cultured BT325 and MDA-MB-231 cells was detected by MTT assay, and cell apoptosis was measured by flow cytometry. RESULTS: The fusion fragment has 805 base pairs, which consists of Kozak consensus sequence, and the sequences encoding alpha-factor signal peptide, EGF, flexible linker, and E4orf4. Recombinant plasmids pAO-EGF-E4orf4, and pAO-3EGF-E4orf4 were obtained, the latter contained 3 expression cassettes. Apparent molecular weight of fusion protein was 20 ku. Immunoblot analysis showed that the fusion protein was immunoreactive with rabbit-anti-human EGF polyclonal antibody. EGF-E4orf4 in high concentrations (5, and 0.5 microg/ml) inhibited growth of BT325 and MDA-MB-231 tumor cells as compared with controls. Apoptosis was induced in 15.4%-28.2% of MDA-MB-231 cells by EGF-E4orf4 at the dosage of 10-25 microg/3 x 10(5) cells. CONCLUSIONS: Fusion protein EGF-E4orf4 may enter cells mediated by EGFR, and thus inhibit growth of tumor cells.

Adenovirus E4 Proteins↗
PubMed1995

Molecular cloning of the alpha 3 chain of human type IX collagen: linkage of the gene COL9A3 to chromosome 20q13.3.

Type IX collagen is composed of three polypeptides derived from the human genes COL9A1, COL9A2, and COL9A3 that assemble to form a mature collagen molecule with the structure alpha 1(IX)alpha 2(IX)alpha 3(IX). We have identified overlapping cDNA and genomic clones that encode for the entire alpha 3 chain of human type IX collagen. Tryptic peptides from the human alpha 3(IX) collagen chain were subjected to N-terminal amino acid sequencing, and a stretch of 124 contiguous amino acids that included the NC1, COL1, and NC2 domains was obtained. Degenerate oligonucleotide primers were designed based on the amino acid sequences of the human tryptic peptides as well as bovine peptides and sequences from chicken cDNA clones. These primers were used to amplify three overlapping PCR products that covered the majority of the human alpha 3(IX) collagen. PCR products were then used to identify overlapping cDNA clones from a human chondrocyte library. A lambda genomic clone was identified that contained the 5'-most exon that encodes the signal peptide to complete the entire structure of the human alpha 3(IX) collagen chain. Genomic amplification identified a single-strand conformational polymorphism in COL1 that was used to map COL9A3 to chromosome 20q13.3 by linkage analysis. The present study completes the structure of human type IX collagen, and linkage for COL9A3 completes the genomic mapping of cartilage collagen genes. These data will greatly assist the genetic screening of families with degenerative cartilage and eye diseases by allowing investigators to screen for a complete set of candidate collagen gene markers.

Amino Acid Sequence↗
PubMed2004

[Construction of phage display antibody library to MCF-7 cells and screening of single-chain antibodies against breast cancer cells].

The aim of this study is to construct a phage display single-chain variable fragment (scFv) library against breast cancer cells and screen the specific antibodies against MCF-7 cells from the library. The BALB/C mice were immunized with MCF-7 cells. Total RNA of spleens was isolated. The heavy-chain (VH) and light-chain variable region genes (VL) of the antibodies were amplified by RT-PCR and joined into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The assembled scFv fragments were cloned into the phagemids(pCANTAB5E) and the recombinant phagemids were used to transform competent E. coli TG1. The transformed TG1 cells were infected by helper phage M13KO7 and the recombinant phagemids were rescued. The scFv fusion proteins were displayed on the surfaces of the recombinant phages. A phage display antibody library of repertoire of 1.2 x 10(6) clones was constructed. The specific antibodies against MCF-7 cells were enriched by 75 times after five rounds of affinity selection. Ten recombinant phages clones that exhibited specific binding to MCF-7 cells were identified. The specificity of those phage clones was analyzed by reactivity against HepG2 cells and Hela cells by ELISA. One of the selected phage clones against MCF-7cells was used to infect E. coli TOP10 to produce the soluble scFv antibodies after induction with IPTG. The strategy of construction and screening of antibody library directed against the whole tumor cells described in this report should be generally applicable to generate tumor cell-specific antibodies.

Animals↗
PubMed2005

[Construction and screening of phage display single chain antibody library against Bursaphelenchus xylophilus cellulase].

A phage display single-chain variable fragment (scFv) library against Bursaphelenchus xylophilus cellulase (BXC) was constructed and used to screen the specific antibodies binding to BXC. The total RNA was extracted from fresh spleens of BALB/C mice immunized with BXC. Gene fragments encoding VH and VL were amplified by RT-PCR and assembled into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The recombinant fragments were cloned into the phagemids (pCANTABSE) and electroporated into E. coli TG1. The recombinant phagemids were rescued by reinfection of helper phage M13K07. The repertoire of the phage display antibody was about 5 x 10(4). The specific antibodies against BXC were obtained after five rounds of affinity selection. The positive phage clone was used to infect E. coli HB2151. SDS-PAGE and western blot analysis showed that the soluble scFv antibodies expressed bound specifically to BXC. The studies laid foundation for quarantine and pathological study of Bursaphelenchus xylophilu.

Animals↗