LacSwitch inducible mammalian expression system in mouse Swiss 3T3 fibroblasts.

Swiss 3T3 fibroblasts were transfected with the provided plasmids of LacSwitch Inducible Mammalian Expression System (Stratagene). Stable transfectants were selected, expanded and characterised. At first, the production of CAT in these cell lines could be induced by IPTG treatment, but the inducibility was lost after a few months in culture in a reproducible manner. Further analysis revealed that the transfectants did not lose the cat gene nor the lac repressor protein. As a result, we conclude that LacSwitch Inducible Mammalian Expression System needs further modification for use in Swiss 3T3 fibroblasts.

AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the alpha2 isoform.

Mammalian AMP-activated protein kinase (AMPK) is the downstream component of a cascade that is activated by cellular stresses associated with ATP depletion. AMPK exists as heterotrimeric alphabetagamma complexes, where the catalytic subunit has two isoforms (alpha1 and alpha2) with different tissue distributions. The budding yeast homologue is the SNF1 kinase complex, which is essential for derepression of glucose-repressed genes, and seems to act by the direct phosphorylation of transcription factors in the nucleus. AMPK complexes containing the alpha2 rather than the alpha1 isoform have a greater dependence on AMP (approx. 5-fold stimulation compared with approx. 2-fold) both in direct allosteric activation and in reactivation by the upstream kinase. We have also examined their subcellular localization by using Western blotting of nuclear preparations, and by using two detection methods in the confocal microscope, i.e. indirect immunofluorescence of endogenous proteins and transfection of DNA species encoding green fluorescent protein-alpha-subunit fusions. By all three methods a significant proportion of alpha2, but not alpha1, is localized in the nucleus. Like SNF1, AMPK-alpha2 complexes could therefore be involved in the direct regulation of gene expression. The observed differences in the regulation of alpha1 and alpha2 complexes by AMP might result in differential responses to ATP depletion in distinct cellular and subcellular locations.

Protein tyrosine phosphatase gene expression analysis in Swiss 3T3 fibroblasts.

The aim of this study was to identify protein tyrosine phosphatases (PTPs) expressed in Swiss 3T3 fibroblasts and to examine their expression levels as well as to characterize quantitative aspects of RT-PCR based on degenerate deoxyoligonucleotides. By using an RT-PCR assay based on degenerate deoxyoligonucleotide primers, expression of mRNAs for two cytoplasmic- and six transmembrane-type PTPs in Swiss 3T3 cells was detected. The sequences of two of them are new. Among nine analyzed PTPs expressed to widely varied extends, only three have mRNA levels high enough to be seen on Northern blots with 10 microg of total RNA per lane. The frequencies with which the examined PTPs are represented among the PCR amplification products, correlate stronger with the primer fidelity, defined as the number of mismatches between the primer- and the cDNA target-sequences, rather than with the PTP expression levels. In conclusion, an RT-PCR assay based on degenerate primers can be successfully used to sample the expressed PTPs and to identify new members of this gene family. However, reliable quantification of their mRNA levels can only be achieved using the classical approaches, like Northern, RNase protection assay or nondegenerate quantitative RT-PCR.

Mouse VH7183 recombination signal sequences mediate recombination more frequently than those of VHJ558.

Mouse VH gene segments are conventionally classified into 13 families on the basis of sequence similarity. The 7183 family lies close to the 3' end of the locus and is preferentially used in BALB/c mice; J558, the largest family, lies close to the 5' end of the VH stretch and is preferentially used in C57BL/6 mice. To investigate whether differential effectiveness of the RSSs in the two families might contribute to the overusage of 7183 in the primary repertoire of BALB/c, we constructed recombination substrates in which the recombination signal sequences (RSSs) of VH segments 7183 and J558 compete with each other for a single RSS, DFL16.1, after transfection into two transformed cell lines derived from C57BL/6 and two cell lines from BALB/c mice. In both strains, the 7183 RSS was found to be preferentially used (83%). Thus, the 7183 RSS mediates recombination more frequently than does that of J558, and this preference must thereby influence the primary repertoire, but the strain difference cannot be accounted for by a difference in the RSSs.

mRNA expression of two transmembrane protein tyrosine phosphatases is modulated by growth factors and growth arrest in 3T3 fibroblasts.

Changes in mRNA expression are physiological regulatory mechanisms and frequently deliver important information regarding functions of corresponding gene products. We investigated changes of abundantly expressed mRNAs of two transmembrane protein tyrosine phosphatases, LRP (leukocyte common antigen-related phosphatase) and mRPTP-sigma. The LRP mRNA expression was modulated by platelet derived growth factor (PDGF) treatment and seems to be regulated by PDGF receptor kinase. The expression of mRPTP-sigma mRNA was low in actively cycling cells, like those in the exponential phase of growth or those treated with different growth factors. In cells whose growth was arrested by contact inhibition at high cell density or by serum starvation at low cell density mRPTP-sigma mRNA level increased. The possible implications of these mRNA expression patterns are discussed.

Overusage of mouse DH gene segment, DFL16.1, is strain-dependent and determined by cis-acting elements.

The DJH structure is of particular importance for diversity in the immunoglobulin heavy chain because it encodes most of CDR3. Here, we investigate mechanisms responsible for generating the DJH structure. We found DFL16.1 was used at a high frequency in normal and transformed pre-B cells (fetal liver > 50%, A-MuLV lines approximately equal to 25%). One DFL16.1JH1 structure was found repeatedly and was also present in DJH and VDJH databases, suggesting this structure may be conserved in the primary repertoire. Genetic analysis demonstrated that C57BL/6 mice use DFL16.1 in DJH structures more frequently than BALB/c. Examination of individual alleles in (C57BL/6 x BALB/c)F1 A-MuLV cell lines revealed that the C57BL/6-derived allele used DFL16.1 twice as often as the BALB/c. This result indicates that part of the mechanism ensuring overusage of DFL16.1 gene segments is cis-acting.

Measurement of recombinase activity in a set of related Abelson murine leukemic virus pre-B cell lines: DJ/DJ lines have more recombinase activity than do VDJ/VDJ lines.

The VDJ recombination potential of a number of Abelson murine leukemic virus transformed fetal liver cell lines derived from (C57BL/6 x BALB/c) F1 mice was measured. The specific developmental stage of each line was determined using Southern blot analysis to ascertain their rearrangement status at the immunoglobulin heavy chain locus (DJ/DJ, VDJ/DJ or VDJ/VDJ). It was observed that DNA from DJ/DJ lines gave many more 'subhaploid' bands hybridizing with JH than did DNA from VDJ/VDJ lines. While the lack of appropriate substrate (VDJ/VDJ lines have exhausted the normal IgH substrate) contributes to the decrease in 'subhaploid bands', this result indicates that the rate of ongoing immunoglobulin heavy chain gene rearrangement was higher in the DJ/DJ lines. However, when the lines were examined using the assay developed by Hesse et al. (4) to measure VDJ recombinase activity, it was found that although all lines had recombinase activity, the DJ/DJ lines had four times more VDJ recombinase activity than did the VDJ/VDJ lines.