We have developed efficient methodologies for construction and expression of comprehensive phage display libraries of murine Fab antibody fragments in E. coli cells. Our methods optimize several ...critical steps of the polymerase chain reaction (PCR) amplification of transcripts of the re-arranged immunoglobulin genes and of their subsequent assembly and expression: Firstly, we have designed exhaustive sets of PCR primers of low degeneracy for the amplification of transcripts of the Fab region of the heavy and light-chain genes. These primers proved effective in amplification of Fab gene fragments from a large panel of hybridoma cell lines of different specificity and family sub-type. Secondly, we have developed a 'jumping PCR' technique that effectively assembled and recombined the amplified heavy and light-chain gene fragments into a bi-cistronic operon. Thirdly, we have constructed expression vectors for insertion of the combinatorial Fab gene-cassette in fusion with a truncated version of the phage surface protein, gIIIp. The heavy chain and the light chain-gIII fusion are transcribed as a polycistronic mRNA from the lacZ promoter and efficient transcriptional control is provided by wildtype lacI present on the vector. The utility of the system was demonstrated by isolating several antigen-binding clones from hybridomas and libraries made from immunized mice.
We have identified and characterized the full set of spliceosomal small nuclear RNAs (snRNAS; U1, U2, U4, U5 and U6) from the ciliated protozoan Tetrahymena thermophila. With the exception of U4 ...snRNA, the sizes of the T. thermophila snRNAs are closely similar to their metazoan homologues. The T. thermophila snRNAs all have unique 5' ends, which start with an adenine residue. In contrast, with the exception of U6, their 3' ends show some size heterogeneity. The primary sequences of the T. thermophila snRNAs contain the sequence motifs shown, or proposed, to be of functional importance in other organisms. Furthermore, secondary structures closely similar to phylogenetically proven models can be inferred from the T. thermophila data. Analysis of the snRNA sequences identifies three potential snRNAs-snRNA base-pairing interactions, all of which are consistent with available phylogenetic data. Two of these occur between U2 and U6, whereas the third occurs between U1 and U2. The proposed interactions locate the intron 5' splice-site close to the intron branch-site nucleotide as well as to the most highly conserved domain of U6. We envisage that these interactions may facilitate the first step of pre-mRNA splicing.
The active sites of the enzyme phenylalanine ammonia-lyase (Pal) from Rhodosporidium toruloides contains a dehydroalanine residue that is believed to be essential for catalytic activity. Furthermore, ...the dehydroalanine is believed to be added post-translationally as part of a prosthetic group covalently attached to the enzyme. Perhaps for this reason no attempts to produce Pal in foreign host cells have been reported. We have inserted the entire uninterupted pal gene from R. toruloides into the Escherichia coli expression vector pKK 223-3. E. coli cells containing this vector synthesize a protein of the expected size, and extracts prepared from these cells contain a Pal-like activity. The potential implications of this finding are discussed.
PCR clamping Orum, H
Current Issues in Molecular Biology
2, Številka:
1
Journal Article
Recenzirano
Odprti dostop
An efficient, PCR based method for the selective amplification of DNA target sequences that differs by a single base pair is described. The method utilises the high affinity and specificity of PNA ...for their complementary nucleic acids and that PNA cannot function as primers for DNA polymerases.
Locked nucleic acids (LNAs) are a family of conformationally locked oligonucleotide analogs inducing unprecedented binding affinity towards DNA/RNA target sequences. Importantly, by virtue of the ...structural resemblance of LNAs to natural nucleic acid monomers, a combination of LNA chemistry with other oligonucleotide chemistries can be exploited to fine-tune the properties towards optimized antisense drug development and target validation technology. The first promising antisense results from experiments with LNA in living animals are described.
Locked nucleic acid (β-D-LNA) monomers are conformationally restricted nucleotides bearing a methylene 2′-O, 4′-C linkage that have an unprecedented high affinity for matching DNA or RNA. In this ...study, we compared the in vitro and in vivo properties of four different LNAs, β-D-amino LNA (amino-LNA), β-D-thio LNA (thio-LNA), β-D-LNA (LNA), and its stereoisomer α-L-LNA in an antisense oligonucleotide (ODN). A well-known antisense ODN design against H-Ras was modified at the 5′- and 3′-ends with the different LNA analogues (LNA-DNA-LNA gapmer design). The resulting gapmers were tested in cancer-cell cultures and in a nude-mouse model bearing prostate tumor xenografts. The efficacy in target knockdown, the biodistribution, and the ability to inhibit tumor growth were measured. All anti H-Ras ODNs were very efficient in H-Ras mRNA knockdown in vitro, reaching maximum effect at concentrations below 5 nM. Moreover, the anti-H-Ras ODN containing α-L-LNA had clearly the highest efficacy in H-Ras knockdown. All LNA types displayed a great stability in serum. ODNs containing amino-LNA showed an increased uptake by heart, liver, and lungs as compared to the other LNA types. Both α-L-LNA and LNA gapmer ODNs had a high efficacy of tumor-growth inhibition and were nontoxic at the tested dosages. Remarkably, in vivo tumor-growth inhibition could be observed at dosages as low as 0.5 mg kg⁻¹ per day. These results indicate that α-L-LNA is a very promising member of the family of LNA analogues in antisense applications.
PDF
