Biomedical subjects

R V Trask

Publications and source records attributed to R V Trask.

5 recordsLinked to original sources

PubMed1992

The human M creatine kinase gene enhancer contains multiple functional interacting domains.

Cis-elements (-933 to -641) upstream of the human M creatine kinase gene cap site contain an enhancer that confers developmental and tissue-specific expression to the chloramphenicol acetyltransferase gene in C2C12 myogenic cells transfected in culture. Division of the enhancer at -770 into a 5' fragment that includes the MyoD binding sites (-933 to -770) and a 3' fragment that includes the MEF-2 binding site (-770 to -641) resulted in two subfragments that showed minimal activity but in combination interacted in a position- and orientation-independent fashion to enhance activity of the SV40 promoter in transient transfection experiments. A 5' enhancer construct (-877 to -832) including only one (the low affinity) MyoD binding site was active when present in multiple copies. In contrast, a 3' enhancer construct (-749 to -732) including the MEF-2 binding site was inactive even when present in multiple copies. However, if the 5' construct was extended to include the high-affinity MyoD binding site (-877 to -803) the 5' and 3' constructs interacted in a position- and orientation-independent fashion to activate the SV40 promoter. Thus, the human M creatine kinase enhancer comprises multiple functional interacting domains.

3T3 Cells↗

PubMed1991

Multiple positive and negative elements regulate human brain creatine kinase gene expression.

We characterized the developmental expression of the brain creatine kinase (BCK) gene in the C2C12 myogenic cell line with the use of isoenzyme, Western blot, and Northern blot analyses. The results show that both BCK subunit protein and mRNA are upregulated early in myogenesis, and then downregulated in fully differentiated myotubes. To characterize the transcriptional regulatory mechanisms, a chimeric construct containing 1.2 kilobase pairs of 5'-flanking DNA from the human BCK gene placed upstream of the chloramphenicol acetyltransferase gene in the promoterless plasmid pSVOCAT was transiently transfected into C2C12 cells. In myoblasts and differentiating myotubes, the time course of expression of the constructs paralleled that of endogenous BCK mRNA. Additional constructs prepared by deleting 5'-flanking DNA were also transfected into C2C12 cells. All constructs were preferentially expressed in myoblasts relative to myotubes with absolute levels of expression increasing with deletion of 5'-flanking DNA. In nonmyogenic cells expression of the plasmids also increased with deletion of 5'-flanking DNA. An element from -1150 to -388 was isolated and found to be capable of suppressing expression of the BCK promoter and of heterologous promoters independent of orientation and position and hence to function as a silencer. Thus, BCK expression is mediated by sequences contained in the 5'-flanking DNA, including negative elements active in both C2C12 cells and nonmyogenic cells and elements that mediate the developmental expression of the BCK gene in C2C12 myogenic cells.

3T3 Cells↗

PubMed1991

Regulation of expression of M, B, and mitochondrial creatine kinase mRNAs in the left ventricle after pressure overload in rats.

Pressure overload of the left ventricle induces synthesis of creatine kinase isoenzymes. To determine whether this response is associated with an altered pattern of creatine kinase gene expression, we induced arterial hypertension in rats by suprarenal aortic banding. After 4 days, left ventricular myocardium from hypertensive (n = 7) and normotensive, sham-operated (n = 5) rats was analyzed for isoenzyme activities by chromatography; M and B creatine kinase subunit protein by Western blot; and M, B, and mitochondrial creatine kinase mRNA by Northern blot. Although total creatine kinase activity increased in hypertensive (1,096 +/- 214 IU/g left ventricle) compared with normotensive rats (648 +/- 81 IU/g left ventricle, p less than 0.01), the relative proportions of the cytoplasmic and mitochondrial isoenzymes did not change. The mass of M and B subunits increased 1.9- and 2.7-fold, respectively, in hypertensive compared with control rats. Similarly, the mRNA for M and B subunits as well as mitochondrial creatine kinase increased 2.6-, 1.6-, and 1.8-fold, respectively, in hypertensive rats compared with control rats. Thus, increased energy requirements in acute pressure overload are met by generalized induction of creatine kinase mRNA and subunit protein and not by an isoenzyme switch.

Animals↗

PubMed1990

Tissue-specific distribution and developmental regulation of M and B creatine kinase mRNAs.

To characterize the tissue-specific distribution and developmentally regulated expression of M and B creatine kinase mRNA in rats, total cellular RNA was isolated from adult rat tissues and from skeletal muscle, heart, brain and intestine at selected stages of development. Northern blots were prepared and hybridized with M and B subunit-specific probes derived from the 3'-untranslated region. M creatine kinase mRNA was expressed abundantly in heart and skeletal muscle, and less abundantly in lung. B creatine kinase mRNA was found in all tissues examined except liver and was abundant in brain, heart and intestine. The developmentally regulated expression of M and B creatine kinase mRNA was determined in skeletal muscle, heart, brain and intestine. The developmental program of B creatine kinase mRNA was different for each tissue examined. During development, M creatine kinase mRNA was up-regulated in both heart and skeletal muscle with a different regulatory program. This resulted in replacement of B mRNA by M mRNA as the predominant species at an earlier developmental stage in heart when compared to skeletal muscle.

Animals↗

PubMed1988

Developmental regulation and tissue-specific expression of the human muscle creatine kinase gene.

To define mechanisms regulating expression of M creatine kinase, the human gene including 5'-flanking DNA was cloned, characterized, and partially sequenced. The gene contains 8 exons interrupted by 7 introns spanning 17.5 kilobase pairs of DNA. The intron-exon splice sites were identified and conform to the GT-AG consensus rule. The TATA and CAAT boxes are located at positions -31 and -56 upstream of the transcription start site as determined by primer extension. The 5'-untranslated region is interrupted with the translation start codon located in the second exon. To determine whether sequences within the 5'-upstream DNA confer tissue-specific expression and developmental regulation, constructs containing 2620 base pairs of human M creatine kinase 5'-flanking DNA fused upstream of the chloramphenicol acetyltransferase gene in the promoterless plasmid pSVO-CAT were transfected into cultured C2C12 myoblasts. There was 17-fold induction of chloramphenicol acetyltransferase activity during differentiation as C2C12 myoblasts fused to form myotubes. The M creatine kinase fusion construct was not expressed in transfected nonmuscle cell lines, COS-7 and NIH/3T3. Thus, cis-acting sequences within 2620 base pairs of the cap site are sufficient to direct developmental regulation and tissue-specific expression of the human M creatine kinase gene.

Base Sequence↗