Phorbol esters selectively and reversibly inhibit a subset of myofibrillar genes responsible for the ongoing differentiation program of chick skeletal myotubes.

Abstract

Phorbol esters selectively and reversibly disassemble the contractile apparatus of cultured skeletal muscle as well as inhibit the synthesis of many contractile proteins without inhibiting that of housekeeping proteins. We now demonstrate that phorbol esters reversibly decrease the mRNA levels of at least six myofibrillar genes: myosin heavy chain, myosin light chain 1/3, myosin light chain 2, cardiac and skeletal a-actin, and skeletal troponin T. The steady-state message levels decrease 50-to 100-fold after 48 h of exposure to phorbol esters. These decreases can be attributed at least in part to decreases in transcription rates. For at least two genes, cardiac and skeletal a-actin, some of the decreases are the result of increased mRNA turnover. In contrast, the cardiac troponin T steady-state message level does not change, and its transcription rate decreases only transiently upon exposure to phorbol esters. Phorbol esters do not decrease the expression of the housekeeping genes, a-tubulin, 0-actin, and -y-actin. Phorbol esters do not decrease the steady-state message levels of MyoDl, a gene known to be important in the activation of many skeletal muscle-specific genes. Cycloheximide blocks the phorbol ester-induced decreases in transcription, message stability, and the resulting steady-state message level but does not block the tetradecanoyl phorbol acetate-induced rapid disassembly of the I-Z-I complexes. These results suggests a common mechanism for the regulation of many myofibrillar genes independent of MyoDl mRNA levels, independent of housekeeping genes, but dependent on protein synthesis.

Antibody repertoire development in fetal and neonatal piglets. XXII. λ rearrangement precedes κ rearrangement during B‐cell lymphogenesis in swine

“…β‐actin is a relatively stable cytoskeletal protein generally thought to be present at some level in most cells, although there may be differences among cell types . Our data are derived from tissues comprised of many cell types, so β‐actin was used only as an internal control for positive transcriptional activity …”

“…70 Our data are derived from tissues comprised of many cell types, so b-actin was used only as an internal control for positive transcriptional activity. [71][72][73]…”

Housekeeping genes as internal standards: use and limits

“…The most often considered and used housekeeping genes are those for albumin (for hepatocytes) (Goldsworthy et al, 1993), b-, g-actins (Choi et al, 1991;Wei et al, 1997), cyclophilin (Bjarnason et al, 1998;Jaschke et al, 1998), G3PDH (Petersen et al, 1990;Tang et al, 1996), a-, b-tubulins (Choi et al, 1991;Serels et al, 1998), hypoxantine phosphoribosyltransferase (HRPT) (Marten et al, 1994;Foss et al, 1998), L32 for other cell types (Lemay et al, 1996;Wu et al, 1999) or 18S, 28S rRNA (Finnegan et al, 1993;Bhatia et al, 1994). The essential functions of these molecules are variable (Table 1).…”

Selection and validation of endogenous reference genes using a high throughput approach

“…Thus, endogenous reference genes are used as common denominator in biological fractions where the expression of a test gene is described as the relative ratio over an arbitrarily selected internal control presumed to be stably expressed in all circumstances relevant to the experiment [1-3]. Most frequently, glyceraldehydes-3-phosphate dehydrogenase (GAPDH) [4,5], albumin (for hepatocytes) [6], β-, γ-actins [7,8], cyclophilin [9,10], α-, β-tubulins [7,11], hypoxantine phosphoribosyltransferase (HRPT) [12,13], L32 [14,15] and 18S, 28S ribosomal RNA (rRNA) [16-18] have been used as endogenous reference genes. Depending upon the experimental design, endogenous reference genes have been used individually or in combination for Northern blot analysis, reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) analysis [19,20].…”