This study compares a number of parameters that are important in the ligation of the polymerase chain reaction-amplified DNA inserts into plasmid vectors and their efficient transformation to bacterial cells. The parameters covered were: T4 polynucleotide kinase treatment followed by either the large fragment of E. coli DNA polymerase or T4 DNA polymerase reactions, the amount of T4 DNA ligase, temperature and duration of ligation, molar ratio of insert to vector as well as the total DNA concentration. The results show that the T4 polynucleotide kinase-treated group without further enzymatic manipulation, at an insert to vector ratio of 3:1 gave the highest recombination efficiency when 10 μg/ml DNA and 20 units T4 DNA ligase were applied for ligation for 12 h at 4°C.
Model organisms are essential to study the genetic basis of human diseases. Transgenic mammalian models, especially genetic knock-out mice have catalysed the progress in this area. To continue the advancement, further sophisticated and refined models are crucially needed to study the genetic basis and manifestations of numerous human diseases. Coinciding with the start of the new era of post-genomic research, new tools for establishment of transgenesis, such as nuclear transfer and gene targeting in somatic cells, have become available, offering a unique opportunity for the generation of transgenic animal models. The new technology provides important tools for comparative functional genomics to promote the interpretation and increase the practical value of the data generated in numerous mouse models. This paper discusses the state-of-the-art of the nuclear replacement technology and presents future perspectives.
A gene for ribosomal protein P0 that belongs to the family of ribosomal P proteins was isolated from a Neurospora crassa cDNA library, using polyclonal antibodies against recombinant P0 protein from Saccharomyces cerevisiae. This is the first gene for ribosomal P0 protein to be cloned from filamentous fungi. The derived P0 protein sequence has a strong homology to other eukaryotic P0 proteins; yet, there is a notable alteration in the conservative C-terminal region, placing this protein among the unique sequences from protozoan parasites.
Rab7 is a small GTPase that plays a crucial role in the regulation of transport from early to late endosomes and lysosomes, phagosome maturation and in lysosomal biogenesis in mammalian cells. It contains conserved and unique sequence elements that mediate its function. Two Rab7 genes, Rab7a (703 bp) and Rab7b (707 bp) were identified in the unicellular eukaryote Paramecium by PCR amplification. They contain three short introns of different lengths (28-32 bp) and sequence located at identical positions in both genes. The presence of two Rab7 genes in the Paramecium genome was confirmed by Southern hybridization analysis performed with six different restriction enzymes. Expression of both genes was assessed by Northern blot and RT-PCR. Two transcripts of 1.8 and 2.2 kb were identified by hybridization analysis. The cloned complementary DNAs, both of 618 nucleotides in length, encode polypeptides of 206 amino acids that are 97.6% identical and differ in their C-termini. The predicted protein sequences of Rab7a and Rab7b contain all characteristic domains essential for Rab function: the effector domain (YRATVGADF) and four GTP-binding consensus sequences (GDSGVGKT, WDTAGQ, NKLD, SAK) as well as the prenylation motif (-CC) at the C-terminus indispensable for Rab binding to the membrane. Similarity searches revealed 81.6-82.1% homology of Paramecium Rab7 isoforms to human Rab7 and a lack of an insert typical for the Kinetoplastida - the species that appeared earlier in evolution. Paramecium is the first free-living lower eukaryote in which homologues of Rab7 have been identified that exhibit features similar to those of mammalian Rab7.
β-mannosidase (EC 3.2.1.25, MANB) dissects the non-reducing end of N-linked mannose moieties of glycoproteins in eukaryotic cells. The human β-mannosidase gene was amplified by RT-PCR, cloned and sequenced. The DNA sequence was compared with reported human β-mannosidase DNA sequence and sixteen nucleotide differences were found. The deduced amino-acid sequence showed that seven codons coded the same amino acids and nine codons coded different amino acids with reference to nucleotide substitution positions but did not affect recombinant MANB enzyme activity. No splice mutation was observed after comparison with reported MANB DNA sequences. A 75% homology of deduced amino-acid sequence was observed with mouse, goat and bovine β-mannosidase amino-acid sequences. The cloned β-mannosidase gene was subcloned into pET22b+ and pET28a+ expression vectors to transform the BL21-codon plus cells for expression of recombinant MAN22 and MAN28 enzymes, respectively. The optimized conditions for overexpression of recombinant β-mannosidase enzyme were induction with 1 mM IPTG for 12 h at 37°C. The expressed β-mannosidase enzyme was purified to homogeneity by a combination of DEAE-ion exchange and size exclusion chromatography. The molecular mass of MAN22 and MAN28 enzymes is 97 kDa by SDS/PAGE and is confirmed by western blot analysis. The recombinant enzymes are active at 37°C and at pH 5.0 and showed activity with p-nitrophenyl-β-d-mannopyranoside and not with p-nitrophenyl-α-d-mannopyranoside. The Km value of enzymes was 2.53 mM. The enzyme activity was inhibited by Zn2+, Co2+, Cu2+, Pb2+, Ag1+, iodoacetate, SDS, DMF, DMSO and ethanol. Fe3+, Ca2+ Mg2+, Mn2+, Triton X-100 and PMSF did not inhibit the enzyme activity. Northern blot analysis showed a transcript of about 3.7 kb in all cells and tissues studied. This is the first report on the expression and characterization of recombinant human MANB enzyme.
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.