A cell- and developmental stage-specific promoter drives the expression of a truncated c-kit protein during mouse spermatid elongation.
In the postnatal testis, the c-kit transmembrane tyrosine-kinase receptor is expressed in type A spermatogonia, and its transcription ceases at the meiotic phase of spermatogenesis. Alternative, shorter c-kit transcripts are expressed in post-meiotic germ cells. These transcripts should encode a truncated version of the c-kit protein, lacking the extracellular, the transmembrane and part of the intracellular tyrosine-kinase domains. The 5' end of the alternative c-kit transcripts maps within an intron of the mouse c-kit gene. We now show that this intron contains a promoter active in nuclear extracts of round spermatids, and that two discrete sequences upstream of the transcriptional start site bind spermatid-specific nuclear factors. Deletion of both these sequences abolishes activity of the promoter in vitro. We have also established that this promoter is functional in vivo, in a tissue-and cell-specific fashion, since intronic sequences drive the expression of the E. coli lacZ reporter gene in transgenic mice specifically in the testis. Transgene expression is confined to haploid germ cells of seminiferous tubules, starting from spermatids at step 9, and disappearing at step 13, indicating that cryptic promoter within the 16th intron of the mouse c-kit gene is active in a short temporal window at the end of the transcriptional phase of spermiogenesis. In agreement with these data, western blot experiments using an antibody directed against the carboxy-terminal portion of the mouse c-kit protein showed that a polypeptide, of the size predicted by the open reading frame of the spermatid-specific c-kit cDNA, accumulates in the latest stages of spermatogenesis and in epididymal spermatozoa. An immunoreactive protein of the same size can be produced in both eukaryotic and prokaryotic artificial expression systems.
Albanesi
C
C
Dipartimento di Sanita' Pubblica e Biologia Cellulare, Universita' di Roma Tor Vergata, Rome, Italy.
Geremia
R
R
Giorgio
M
M
Dolci
S
S
Sette
C
C
Rossi
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D017930:Genes, Reporter; D006238:Haploidy; D007438:Introns / Q000235:genetics; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics*; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D017384:Sequence Deletion; D013087:Spermatids / Q000502:physiology; D013091:Spermatogenesis / Q000235:genetics*; D013737:Testis / Q000737:chemistry / Q000166:cytology; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D004247:DNA; D019009:Proto-Oncogene Proteins c-kit; D001616:beta-Galactosidase