Protamines are small arginine-rich proteins that package DNA in spermatozoa. The mouse protamine 1 (Prm-1) gene is transcribed exclusively in post-meiotic spermatids. To identify elements in the Prim-promoter required for spermatid-specific transcription, we generated transgenic mice by microin'ection of transgenes containing Prm-l 5' flanking sequences with 5' truncations or internal deletions of conserved sequences linked to a marked Prm-1 gene. We also tested Prm-l promoter regions with a heterologous human growth hormone reporter gene. We conclude that a 113-bp region can direct spermatid-specific transcription and we have defined sequences within this region that are essential for proper function. These results will facilitate the isolation and characterization of transcription factors essential for postmeiotic gene expression.Spermatogenesis is the sequence of developmental events by which spermatogonial stem cells give rise to functional spermatozoa. During this developmental process, spermatogonial stem cells proliferate through several mitotic divisions followed by two meiotic divisions resulting in haploid round spermatids. During spermiogenesis, the spermatids undergo extensive cytological and biochemical changes leading to the formation of mature spermatozoa (for review, see ref.

The objective of this study was to determine the consequences of disrupting spermatozoal chromatin condensation on spermatozoal development and function. The avian protamine, galline, was targeted to spermatids of transgenic mice using the mouse protamine 1 gene promoter. Three transgenic mouse lines were established that expressed galline mRNA at 65%, 120%, and 185% of the level found in rooster testis. Galline mRNA accumulated in round spermatids to levels similar to that of mouse protamine and, as with the mammalian counterpart, translation was delayed until the elongating spermatid stage. Protein gels revealed that galline accumulated in mature spermatozoa whereas mouse protamines were reduced, suggesting that galline competes with protamines for binding to spermatozoal DNA. Acridine orange binding analysis indicated that DNA of the transgenic spermatozoa was not as tightly packed as that of controls. This was corroborated by electron microscopy, which revealed disruption of the normal dense chromatin structure of spermatozoal heads. Despite these perturbations of chromatin condensation, the transgenic spermatozoa were functionally normal, as the majority of transgenic mice had normal fertility. However, in mice that expressed excessive galline, there was a gradual destruction of seminiferous tubules leading to infertility. Our findings suggest that very precise packaging of DNA in germ cells may not be essential for subsequent unpackaging in the pronucleus of fertilized eggs and for subsequent normal development of the embryo.

Thymidylate synthase (TS) mRNA content increases about 20-fold when growth-stimulated mouse cells progress from the G0/G1 phase into the S phase of the cell cycle. Previous studies, using a cell line in which the TS gene is amplified (LU3-7), indicated that transcriptional initiation as well as polyadenylation of the mRNA occur at several locations in unsynchronized cells. In the present study, we have used S1 nuclease protection assays to analyze the possible significance of the multiple transcriptional initiation and polyadenylation sites. We found that the same pattern of 5' and 3' termini were detected with RNA isolated from the overproducing cells as with RNA isolated from the parental mouse 3T6 cell line, demonstrating that the heterogeneous termini are not a consequence of gene amplification. There was no change in the pattern of 5' or 3' termini with either cell line during the progression from G1 phase through S phase in serum-stimulated cells. Therefore, the increase in TS mRNA content is not the result of differential utilization of the various transcriptional initiation or polyadenylation sites. Analyses of poly(A)- deficient cytoplasmic TS RNA showed that the 5' termini were the same as those found in poly(A) + mRNA. However, the 3' termini were extremely heterogeneous in length. Although some of the poly(A)- deficient RNA extended beyond the normal site of polyadenylation, most of it was shorter than full-length TS mRNA.

The mouse thymidylate synthase (TS; EC 2.1.1.45) mRNA is unusual in that the poly(A) tail is added at the translation stop codon. To determine the sequence requirements for 3' processing of this mRNA, we constructed TS minigenes with deletion and point mutations in potential regulatory sequences. The minigenes were transiently transfected into cultured cells and the effect on 3' processing was determined by Si nuclease protection assays. These analyses revealed that at least two elements are required for efficient polyadenylylation at the stop codon. The first is an upstream AUUAAA sequence. When this was changed to AUCAAA, polyadenylylation at the stop codon was blocked. However, when it was changed to the canonical AAUAAA hexanucleotide, the amount of TS mRNA increased severalfold. The second element is a stretch of 14 consecutive uridylate residues 32 nucleotides downstream of the stop codon. This U-rich region is absent from the human TS gene, which explains why the human TS mRNA is not polyadenylylated at the stop codon even though the two genes are otherwise almost identical through this region. The most surprising observation was that the U-rich region corresponds to the 3' end of a 360-nucleotide mouse Ll repetitive element that was inserted in opposite orientation to the gene more than 5 million years ago. Thus the polyadenylylation signal of the present mouse TS gene was created by the transposition ofa repetitive element downstream of a cryptic polyadenylylation signal. the final A of the UAA stop codon is added by poly(A) polymerase. Sequences that conform to polyadenylylation consensus elements are present in the vicinity of the stop codon of the TS gene. These include a variant hexanucleotide, AUUAAA, in the coding region, a G+U-rich sequence immediately downstream of the stop codon, and 14 consecutive uridylate residues beginning 32 nucleotides downstream of the stop codon (10). The human and mouse TS genes are very similar across the coding region and differ at only one position between the AUUAAA and G+U-rich sequences (see Fig. 1B) (10, 12). In spite of this, the human TS mRNA is polyadenylylated not at the stop codon but rather 500 nucleotides downstream (12). It was suggested that the human TS mRNA was not polyadenylylated at the stop codon because it lacked the oligo(U) region (10).In this paper, we identify the upstream and downstream sequences that are important for the polyadenylylation of mouse TS mRNA. We show that variations in these sequences lead to large changes in the efficiency of utilization of the major and minor polyadenylylation sites. The oligo(U) region was found to be essential for directing efficient polyadenylylation of TS mRNA at the stop codon. The oligo(U) region and sequences distal to it correspond to the 3' end of a mouse Li repetitive element that was inserted in opposite orientation relative to the direction of transcription of the TS gene.

No abstract