Muscle coding sequences and their regulation during myogenesis : cloning of muscle actin cDNA probes

Abstract

(Buckingham, 1977). Experiments using actinomycin D suggested that mRNA coding for muscle protein accumulates just prior to cell fusion (e.g. Yaffe and Dym, 1973 (Glover, 1977). We have employed this technique to isolate muscle coding sequences from heterogeneous fractions of mouse muscle mRNA. The protocol is summarized in figure 2. Poly A + RNA was separated on low salt/sucrose gradients to obtain partially enriched fractions for the myosin light chains, the actins, and the myosin heavy chain mRNAs. Although the proteins are very abundant in muscle (20-60 p. 100 total protein), the level of protein synthesis and of the corresponding mRNA is very much lower (1-5 p. 100) which makes purification of the mRNAs difficult (c/f globin mRNA 50-90 p. 100). Once the mixture of double-stranded cDNAs has been integrated into plasmids ( fig. 2) and bacterial clones selected with the expected antibiotic resistance, the major problem is identification of the clones. A preliminary screening was carried out in situ by the technique of Grunstein and Hogness (1975), using 32 P-labelled C DNA from the original RNA fraction and from muscle and nonmuscle RNA to obtain as much information as possible. Clones were then selected and grown in bulk culture, and the plasmid DNA was purified. This DNA was used in hybridization/translation assays with muscle mRNA, either in the hybrid-arrested translation technique (HART) (Paterson et al.,1977) (Minty et al., 1981). The DBM filter technique indicated that this clone cross-hybridizes with muscle and non-muscle actin mRNA. After hybrid-arrested translation and analysis of the products on two-dimensional gels, it appears to affect the translation of (1.-and y-actin mRNAs more than that of (3-actin mRNA. In order to determine which sequence is incorporated into the plasmid, the inserted C DNA segment was isolated and its DNA sequence analyzed by the method of Maxam and Gilbert (1977). The result is shown in figure 3 where it is clear that the clone contains a sequence corresponding to oe-actin. Analysis of this sequence by restriction mapping and partial DNA sequencing indicates that it contains 1 350 nucleotides, including part of the non-coding 3' terminal of the mRNA and lacking a short sequence at the 5' coding end of the messenger (fig. 4)

Correlation of B-FABP and GFAP expression in malignant glioma

“…The following probes were used for hybridization: a 700 bp human B-FABP cDNA insert described in the Results section, a 2.3 kb human GFAP cDNA insert from the American Type Culture Collection, Rockville, MD) (GenBank/EMBL No. M78090), a 1.3 kb HindIII fragment from the ribosomal protein gene rpL32/4A (Dudov and Perry, 1984) and mouse a-actin cDNA (Minty et al,1981).…”

Down‐modulation of c‐kit mRNA and protein expression by erythroid differentiation factor/activin A

“…Amplified 381 bp fragment was cloned into TA cloning vector (Invitrogen Co., San Diego, CA) and identified by DNA sequencing. Murine c-kit, fl-globin [19], fl-actin [20], uroporphyrinogen decarboxylase (UPD) [21] cDNA fragments were electrophoretically purified from agarose gels and labeled by using hexadeoxynncleotide random primers (Amersham International, Buckinghamsire, UK) with [~-32P]dCTP (specific activity 111 TBq/mmol; NEN Research Products, Boston, MA).…”

cDNA recombinant plasmid complementary to mRNAs for light chains 1 and 3 of mouse skeletal muscle myosin.

“…3, lane c), which gives bands similar to, but fainter than, those seen with mouse muscle (Fig. 3, lane d) (5,(35)(36)(37) and throughout evolution (36,37).…”

“…We have, therefore, undertaken the cloning of cDNA probes of the sequences coding for mouse muscle contractile proteins. We have already reported the characterization of a muscle actin plasmid (5). We describe here the cloning and characterization of a recombinant plasmid that hybridizes specifically with the messengers coding for the muscle myosin light chains LC1 and LC3.…”