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Molecular cloning of the human Hand1 gene/cDNA and its tissue-restricted expression in cytotrophoblastic cells and
M Knöfler1, G Meinhardt, R Vasicek
1Department of Obstetrics and Gynecology, University of Vienna, W ahringer Gürtel 18-20, A-1090, Vienna, Austria. martin.knoefle@akh-wien.ac.at
Insights
The human Hand1 gene, a basic helix-loop-helix factor, is cloned and sequenced. Hand1 is primarily expressed in the adult heart, suggesting a role in cardiac function, but not in villous trophoblast development.
Area of Science:
- Molecular biology
- Developmental biology
- Genetics
Background:
- The basic helix-loop-helix (bHLH) factor Hand1 is crucial for heart development in chickens and mice.
- Hand1's role in human embryonic development, particularly in cardiac and trophoblast lineages, requires detailed investigation.
Purpose of the Study:
- To clone and characterize the human Hand1 gene and cDNA.
- To analyze the expression pattern of human Hand1 mRNA in various tissues and cell types.
- To elucidate the potential roles of Hand1 in human cardiac and trophoblast development.
Main Methods:
- Cloning of human Hand1 cDNA from a heart-specific library and genomic DNA from a lambda-DNA library.
- Nucleotide sequencing of the Hand1 cDNA and gene, including exons and introns.
- In vitro transcription-translation assays to analyze Hand1 protein characteristics.
- Southern blot analysis to confirm the presence of the entire Hand1 gene.
- Reverse transcription-polymerase chain reaction (RT-PCR) to determine Hand1 mRNA expression patterns in different human tissues and cell lines.
Main Results:
- The human Hand1 cDNA sequence was determined, encoding a 215 amino acid protein with a conserved bHLH region.
- The Hand1 gene consists of two exons and one intron, with the genomic clone containing the entire gene.
- In vitro studies suggest post-translational modification of the Hand1 protein.
- Hand1 mRNA expression is predominantly detected in the adult heart.
- Hand1 transcripts are absent in villous trophoblast cells but abundant in certain trophoblastic cell lines (Jeg-3, BeWo).
Conclusions:
- Human Hand1 is a cardiac-specific transcription factor potentially involved in adult heart function.
- Hand1 is not essential for the development of villous and extravillous trophoblast lineages.
- Hand1 may play a role in early trophoblast differentiation, as indicated by its expression in specific cell lines.
Abstract:
The basic helix-loop-helix (bHLH) factor Hand1 plays a role in the developing chicken heart and is required for trophoblast giant cell differentiation and cardiac looping of mouse embryonic development. Here, we report the cloning of the human Hand1 cDNA and gene from a heart-specific cDNA library and a genomic lambda-DNA library, respectively. We present the nucleotide sequence of a 1.75kb cDNA clone, encoding the presumptive 215 amino acid human Hand1 protein, and show homology comparison of the conserved bHLH region between different species. In vitro transcription-translation of Hand1 mRNA and analysis of protein size suggest that the Hand1 polypeptide is (post)translationally modified. By Southern blot analysis we demonstrate that the isolated genomic DNA clone harbours the entire Hand1 gene and describe molecular structure and sequences of the two 799 and 938bp exons and the single 1.56kb intron. The expression pattern of the mRNA in different human tissues revealed that Hand1 transcripts are restricted to the heart, suggesting that the protein could be required for cardiac-specific gene transcription and function in adults. Hand1 transcripts were undetectable in a non-tumorigenic villous trophoblast cell line, immunopurified cytotrophoblasts undergoing in vitro differentiation, and first trimester placental tissue, suggesting that the transcription factor is not involved in the development of villous and extravillous trophoblast cell lineages. Hand1 mRNA, however, was abundantly expressed in cytotrophoblastic Jeg-3 and BeWo cells, suggesting that Hand1 could be required for early trophoblast differentiation.