A new troponin T and cDNA clones for 13 different muscle proteins, found by shotgun sequencing

Abstract

Complete amino acid sequences have been established for 19 muscle-related proteins and these proteins are each sufficiently abundant to suggest that their mRNA levels are about 0.4% or higher. Based on these considerations, a simple theoretical analysis shows that clones for most of these proteins can be identified within a complementary DNA library by sequencing cDNA inserts from 150-200 randomly selected clones. This procedure should not only rigorously identify specific clones, but it could also uncover amino acid sequence variants of major muscle proteins such as the troponins. We have determined sequences for about 20,000 nucleotides within 178 randomly selected clones of a rabbit muscle cDNA library, and report here that in addition to finding sequences encoding the two known skeletal muscle isotypes of troponin C, we have discovered sequences encoding two forms of troponin T. Over the region of nucleotide sequence overlap in the troponin T clones, the new isotype diverges significantly from its counterpart. Altogether, clones for 13 of the 19 known muscle-specific proteins were identified, in addition to the clone for the new troponin T isotype.

Comparative sequence analysis of rat, rabbit, and human muscle glycogen phosphorylase cDNAs

“…The predicted amino acid sequence is identical to that previously determined by amino acid sequence analysis [19]. However, portions of the rabbit phosphorylase cDNA vary slightly in nucleotide sequence from cDNAs reported previously by Putney et al [43] suggesting the possibility of allelic variation at this locus. (The differences represent silent changes at positions of leucine-661 and leucine-720).…”

Transcriptional profiling in pearl millet (Pennisetum glaucum L.R. Br.) for identification of differentially expressed drought responsive genes

“…This would substantially increase the chances for identification of rare transcripts involved in drought stress. The concept of using cDNAs as a route to expedite gene discovery was first demonstrated in the early 1980s (Putney et al 1983). Later gene discovery in most plants was done primarily by sample sequencing of expressed sequence tags (ESTs) (Lim et al 1996;Covitz et al 1998).…”

Gene Expression Profile of Human Bone Marrow Stromal Cells: High-Throughput Expressed Sequence Tag Sequencing Analysis

“…Although incomplete and not error-free, ESTs remain an effective means for novel gene discovery and generating biologically informative probes for mapping genes to chromosomes as sequence-tagged sites (STSs), for identifying mutations leading to heritable diseases, and for full-length cDNA cloning [21,26]. The advantages of this approach are as follows: (1) it can be pursued as a relatively inexpensive and rapid way to access many of the expressed genes of a cell or tissue type [29,30]; and (2) with the advent of highthroughput sequencing technology and an increased interest in genome-wide studies, it became clear that ESTs could be generated in sufficient numbers to provide a rapid means of gene discovery [28,31], especially for those searching for human disease genes or constructing physical maps of the human genome [32].Based on the feasibility of EST analysis for gene discovery and pattern configuration in other cell types or organisms [31], we undertook a larger-scale EST project to sequence randomly isolated cDNAs from a HBMSC cDNA library. The FIG.…”