ALTERNATIVE SPLICING OF THE CARDIAC TROPONIN T GENE

  • Lipshultz, Steven E, (PI)

Project: Research project

Description

The goal of this proposal is to elucidate the mechanisms involved in the
generation, by a single contractile protein gene, of multiple mRNAs
encoding different proteins that are tissue-specific and developmentally
regulated. This analysis will concentrate on the elucidation of the
structure of the mammalian cardiac troponin T (TnT) gene and its regulation
during development, in response to hormone stimulation and to work overload
hypertrophy. It is not yet known whether this variety of stimuli are able
to act on the gene in an independent manner, either directly or indirectly,
or whether they all act through a common pathway affecting a unique DNA
sequence in the gene. Moreover, it is not yet known whether the different
genes expressed during development and in response to different stimuli
have common regulatory sequences. The essential role of TnT in striated
muscle contraction relates to its close interaction with tropomyosin and
with the calcium binding protein troponin C in the sarcomere. Other
striated muscle TnT genes utilize alternative splicing of common and
isotype specific exons to generate isoform diversity in response to
developmental and tissue-specific regulation by the production of multiple
mRNAs. RNA splicing defects have been implicated as the mechanism
contributing to several clinically relevant states (ie. Thalassemias).
However, detailed mechanisms of alternative splicing remain obscure. This
proposal will attempt to determine whether the mammalian cardiac TnT gene
utilizes alternative splicing and to understand the mechanism(s) involved
in this process. A three part approach will be followed. First, the cDNA
and genomic clone corresponding to this gene will be isolated and
characterized by differential colony hybridization, Northern blot
hybridization, S1-nuclease analysis and DNA sequencing. Second, the normal
and abnormal patterns of expression of this gene in the myocardium will be
correlated in search for common features that could shed light on its mode
of regulation. Analysis of these clones under different conditions will
occur by S1-nuclease mapping and 5' extension techniques. Finally, the
dissection of the molecular events involved in the regulation of this gene
and of mechanisms of alternative splicing, if it occurs, will be initiated
using a combination of in vivo and in vitro systems including specific gene
constructs.
StatusFinished
Effective start/end date7/1/866/30/91

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

Fingerprint

Troponin T
Alternative Splicing
Genes
Clone Cells
Troponin C
RNA Splicing
Contractile Proteins
Sarcomeres
Tropomyosin
Calcium-Binding Proteins
Thalassemia
Protein C
DNA Sequence Analysis
Exons
Myocardium
Protein Isoforms
Hormones
Gene Expression
Muscles
Proteins

ASJC

  • Medicine(all)