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K C Kleene

Publications and source records attributed to K C Kleene.

At least 37 records · Page 2Linked to original sources
PubMed1989

Mouse transition protein 1 is translationally regulated during the postmeiotic stages of spermatogenesis.

Transition protein 1 (TP1) is a small basic nuclear protein that functions in chromatin condensation during spermatogenesis in mammals. Here, recently identified cDNA clones encoding mouse transition protein 1(mTP1) were used to characterize the expression of the mTP1 mRNA during spermatogenesis. Southern blot analysis demonstrates that there is a single copy of the gene for transition protein 1 in the mouse genome. Northern blot analysis demonstrates that mTP1 mRNA is a polyadenylated mRNA approximately 600 bases long, which is first detected at the round spermatid stage of spermatogenesis. mTP1 mRNA is not detectable in poly(A)+ RNAs isolated from mouse brain, kidney, liver, or thigh muscle. mTP1 mRNA is translationally regulated in that it is first detected in round spermatids, but no protein product is detectable until approximately 3 days later in elongating spermatids. In total cellular RNA isolated from stages in which mTP1 is synthesized, the mTP1 mRNA is present as a heterogeneous class of mRNAs that vary in size from about 480 to 600 bases. The shortened, heterogeneous mTP1 mRNAs are found in the polysome region of sucrose gradients, while the longer, more homogeneous mTP1 mRNAs are present in the postmonosomal fractions.

Animals↗
PubMed1989

Poly(A) shortening accompanies the activation of translation of five mRNAs during spermiogenesis in the mouse.

I have compared the quantity and the length of the poly(A) tracts of five haploid-expressed mRNAs in the polysomal and nonpolysomal fractions of round and elongating spermatids in mice: transition proteins 1 and 2, protamines 1 and 2, and an unidentified mRNA of about 1050 bases. Postmitochondrial supernatants of highly enriched populations of round and elongating spermatids (early and late haploid spermatogenic cells) were sedimented on sucrose gradients, and the size and amount of each mRNA in gradient fractions were analyzed in Northern blots. In round spermatids, all five mRNAs are restricted to the postpolysomal fractions, but in elongating spermatids about 30-40% of each mRNA is associated with the polysomes. The distribution of these mRNAs in sucrose gradients suggests that all five mRNAs are stored in a translationally repressed state in round and early elongating spermatids, and that they become translationally active in middle and late elongating spermatids. The translationally repressed forms of all five mRNAs are long and homogenous in size, whereas the polysomal forms are shorter and more heterogenous due to shortening of their poly(A) tracts. The relationship between translational activity and poly(A) size exemplified by these five mRNAs may be typical of mRNAs which are translationally repressed in round spermatids and translationally active in elongating spermatids.

Animals↗
PubMed1988

Nucleotide sequence of a cDNA clone encoding mouse transition protein 1.

We have determined the nucleotide sequence of cDNA clones encoding mouse transition protein 1 (TP1), a basic nuclear protein involved in nuclear condensation during spermiogenesis. The nucleotide sequence predicts that transition protein 1 in rats and mice differs by only one amino acid. The rate of substitution of nucleotides in the coding region of mouse and rat transition protein 1 mRNA is close to the average of many proteins in rats and mice, and the usage of degenerate codons is typical of the mouse. The identification of this cDNA clone, in conjunction with previous work (Kleene et al. (1983) Dev. Biol. 98, 455-464; Hecht et al. (1986) Exp. Cell Res. 164, 183-190), demonstrates that the mRNA for mouse transition protein 1 accumulates during the haploid phase of spermatogenesis.

Animals↗
PubMed1987

Characterization of a cDNA clone encoding a basic protein, TP2, involved in chromatin condensation during spermiogenesis in the mouse.

A cDNA clone encoding a small cysteine and serine-rich basic protein has been isolated from a mouse testis cDNA library. This cDNA clone encodes the mouse homologue of a protein involved in the initial phases of condensation of chromatin during spermiogenesis in rats, TP2, based on similarities in the sequence of the carboxyl terminus, composition, molecular weight, and electrophoretic mobility. Mouse TP2 can be divided into a highly basic domain comprising about one-third of the polypeptide chain at the carboxyl terminus and a much less basic domain comprising the remaining two-thirds at the amino terminus. The 5' end of the mouse TP2 mRNA contains two in-phase initiation codons both of which may be used generating two polypeptides which differ in length at the amino terminus. Southern blots demonstrate that there is a single copy of the TP2 gene in the mouse genome and Northern blots demonstrate that the polyadenylated TP2 mRNA is present at high and essentially equal levels in early and late haploid cells, and that it is virtually absent from meiotic cells.

Amino Acid Sequence↗
PubMed1987

Synthesis of mouse t complex proteins during haploid stages of spermatogenesis.

We have analyzed the expression, through spermiogenesis, of a series of testicular cell polypeptides encoded by genes within the mouse t complex. Two of these polypeptides, TCP-3 and TCP-7, are synthesized in a testes-specific manner with highest levels of expression during haploid stages of spermatogenesis. A third, TCP-1, is also expressed at highest levels in haploid cells, and expression of this polypeptide continues until the last residual body stage of spermiogenesis. The genes that encode these polypeptides have been correlated with the t phenotype of transmission ratio distortion.

Animals↗
PubMed1987

Translation of mouse testis poly(A)+ mRNAs for testis-specific protein, protamine 1, and the precursor for protamine 2.

Since previous studies have suggested that the mammalian protamine mRNAs are translated poorly in cell-free systems, we directly measured the efficiency of translation of mouse protamine 1 mRNA. We found that mouse testis poly(A)+ mRNA stimulates the synthesis in the wheat germ and reticulocyte cell-free systems of three prominant translation products which can be resolved by electrophoresis through acid urea polyacrylamide gels containing 8 M urea. These translation products have been identified as testis-specific protein, protamine 1, and the precursor to protamine 2 by several criteria, including labeling with amino acids, [35S]cysteine, and [3H]leucine, which are known to be specific to some of these proteins from the nucleotide sequences of recombinant DNAs. Surprisingly, the mobility of the testis-specific protein translation product is slightly reduced and the mobility of both protamine translation products is drastically reduced unless the extracts of cell-free translations are coelectrophoresed with the appropriate carrier. The fraction of [35S]cysteine- labeled protamine 1 translation product was compared with the fraction of testis poly(A)+ mRNA as protamine 1 mRNA which we measured in dot blots with the use of an SP6 RNA polymerase transcript for protamine 1. The results demonstrate that protamine 1 mRNA is translated only slightly less efficiently than the average testis poly(A)+ mRNA.

Animals↗
PubMed1987

Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not.

The nuclei of mouse spermatozoa contain two protamine variants, mouse protamine 1 (mP1) and mouse protamine 2 (mP2). The amino acid sequence predicted from mP1 cDNAs demonstrates that mP1 is a 50-amino-acid protein with strong homology to other mammalian P1 protamines. Nucleotide sequence analysis of independently isolated, overlapping cDNA clones indicated that mP2 is initially synthesized as a precursor protein which is subsequently processed into the spermatozoan form of mP2. The existence of the mP2 precursor was confirmed by amino acid composition and sequence analysis of the largest of a set of four basic proteins isolated from late-step spermatids whose synthesis is coincident with that of mP1. The sequence of the first 10 amino acids of this protein, mP2 precursor 1, exactly matches that predicted from the nucleotide sequence of cDNA and genomic mP2 clones. The amino acid composition of isolated mP2 precursor 1 very closely matches that predicted from the mP2 cDNA nucleotide sequence. Sequence analysis of the amino terminus of isolated mature mP2 identified the final processing point within the mP2 precursor. These studies demonstrated that mP2 is synthesized as a precursor containing 106 amino acids which is processed into the mature, 63-amino-acid form found in spermatozoa.

Amino Acid Sequence↗
PubMed1986

Equations describing the effects of the transient stability of subpopulations of nascent hnRNAs on the kinetics of turnover of hnRNA.

The equations that have been used previously to analyse the rate of decay of hnRNA implicitly assume that nascent hnRNAs are degraded stochastically. This assumption is inconsistent with electron-microscopic studies of transcription cited here, which show that nascent hnRNAs are not degraded during transcription, implying that hnRNA degradation occurs only after termination of transcription and release of the hnRNA from chromatin. Equations are derived describing the accumulation of radioactivity hnRNA during continuous labelling assuming that nascent hnRNAs are stable and that hnRNAs decay with first-order kinetics only after completion of transcription. The effects of the transient stability of nascent hnRNAs on the kinetics of hnRNA turnover can become important when the half-life of the hnRNA is shorter than the time to transcribe an hnRNA from the point of initiation to the point of termination. These equations should prove useful in studies of hnRNA turnover that require a precise description of the labelling kinetics of nascent and completed subpopulations of hnRNA.

Animals↗
PubMed1986

Mapping of haploid expressed genes: genes for both mouse protamines are located on chromosome 16.

Mouse spermatozoa contain two protamines with different amino acid sequences. By hybridizing Southern blots of a series of mouse-hamster somatic cell hybrids containing subsets of mouse chromosomes and a complete set of hamster chromosomes with 32P-labeled cDNAs for each mouse protamine, we assign the two mouse protamine genes to chromosome 16. This report presents the first evidence for chromosomal linkage of two sperm-specific, haploid regulated gene products.

Animals↗
PubMed1985

Nucleotide sequence of a cDNA clone encoding mouse protamine 1.

The nucleotide sequence of a 404-base cDNA encoding the cysteine-rich, tyrosine-containing mouse protamine has been determined. This insert, isolated from a mouse testis cDNA library, encodes a polypeptide of 50 amino acids of which 28 are arginine, 9 are cysteine, and 3 are tyrosine. The insert contains the complete 3' noncoding region of 151 bases and most of the 5' noncoding region. The predicted amino acid sequence of mouse protamine 1 is about 80% homologous to boar protamine and 67% homologous to bull protamine and contains the central, highly basic domain of four arginine clusters found in the trout protamines. The identification of a cDNA clone for a mouse protamine will facilitate studies of the evolution, regulation, and protein-DNA interaction of this nuclear protein unique to haploid spermatogenic cells.

Amino Acid Sequence↗
PubMed1985

Size changes of protamine 1 mRNA provide a molecular marker to monitor spermatogenesis in wild-type and mutant mice.

We utilized a cDNA encoding the cysteine-rich, tyrosine-containing mouse protamine, mouse protamine 1 (MP1), to detect the presence of several classes of differentiating germ cells in testicular extracts from wild-type and male sterile mutant mice. This assay is based on the changes in the poly (A) length of MP1-mRNA during spermatogenesis. Testicular extracts of sexually mature CD-1 mice contain a heterogeneous population of protamine-1 mRNA ranging in length from 450 to 580 nucleotides. When the protamine-1 probe was hybridized to testicular RNA preparations from 16- to 20-day-old animals, no MP1-mRNA was detected. Twenty-four-day-old mice contain only the 580-nucleotide form of MP1-mRNA. This size class of protamine mRNA is also present in purified populations of round spermatids, whereas elongating spermatids and residual bodies contain mRNAs ranging from 450 to 580 nucleotides in length, which are identical in size to those present in the testes of sexually mature animals. When the protamine cDNA probe was used to examine the progression of spermiogenesis in three male sterile mouse mutants, blind sterile (bs), quaking (qk) and testicular feminization (Tfm), the results demonstrated that each mutant is pathologically distinct. Analysis of the bs mutant revealed a diminution in the amount of both size classes of MP1-mRNA, in agreement with the cytological reports of reduced numbers of haploid spermatogenic cells in these animals. The presence of both size classes of protamine mRNA in the qk mutant indicates that germ-cell differentiation has proceeded at least to the step-12 spermatid in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgen-Insensitivity Syndrome↗
PubMed1985

Transcription of similar sets of rare maternal RNAs and rare nuclear RNAs in sea urchin blastulae and adult coelomocytes.

We studied the sequences transcribed in the rare class of hnRNA and the rare maternal RNA set in blastula embryos and a tissue of adult sea urchins, coelomocytes. About 26% of labelled single-copy DNA formed hybrids which bound to hydroxyapatite after three cycles of hybridization with nuclear RNA from blastulae and coelomocytes. This corresponds to transcription of about 50% of the single-copy genome by both cell populations. To compare the rare hnRNA sequences synthesized by blastulae and coelomocytes directly, labelled single-copy DNA was hybridized with blastula nuclear RNA to high RNA C0t, fractionated into sequences complementary and non-complementary to blastula nuclear RNA by chromotography on hydroxyapatite, and then each fraction was rehybridized with nuclear RNA from blastulae and coelomocytes. About 62% of the labelled DNA complementary to blastula nuclear RNA and about 1.5% of the labelled DNA non-complementary to blastula nuclear RNA hybridized with nuclear RNA from both cell populations. Thus, coelomocytes and blastula embryos transcribe essentially the same single-copy sequences in the rare hnRNA class. A probe for the rare maternal RNA set was isolated by hybridizing single-copy DNA with total egg RNA to high RNA C0t. 65-67% of this probe hybridized with whole-cell RNA from eggs, blastulae, plutei and coelomocytes demonstrating that essentially all rare maternal RNAs are present, and presumably transcribed, in blastulae, plutei and coelomocytes.

Animals↗
PubMed1984

Haploid expression of a mouse testis alpha-tubulin gene.

A complementary DNA clone for an alpha-tubulin has been isolated from a mouse testis complementary DNA library. The untranslated 3' end of this complementary DNA is homologous to two RNA transcripts present in postmeiotic cells of the testis but absent from meiotic cells and from several tissues including brain. The temporal expression of this alpha-tubulin complementary DNA provides evidence for the haploid expression of a mammalian structural gene.

Animals↗
PubMed1984

Maternal inheritance of the mouse mitochondrial genome is not mediated by a loss or gross alteration of the paternal mitochondrial DNA or by methylation of the oocyte mitochondrial DNA.

To evaluate whether the absence or modification of paternal mitochondrial DNA or methylation of the oocyte mitochondrial DNA could be the molecular basis for maternal inheritance of mitochondria in mammals, the mitochondrial genome has been analyzed in four meiotic and postmeiotic testicular cell types, and in oocytes from the mouse. All four testicular cell types including spermatozoa contain mitochondrial DNA. Between meiosis and the end of spermatogenesis the number of mitochondrial genomes per haploid genome decreases 8- to 10-fold with spermatozoa containing approximately one copy of the mitochondrial genome per mitochondrion. Restriction enzyme digestions with six different enzymes indicate no gross differences in DNA sequence in the testicular mitochondrial DNA from meiotic cells, early haploid cells, late haploid cells, and spermatozoa. By the criterion of differential digestion with the isoschizomers, MspI and HpaII, the mitochondrial DNA is not differentially methylated during spermatogenesis. No methylation differences were detected in mitochondrial DNA from sperm and oocytes following digestion with seven methylation-sensitive restriction enzymes.

Animals↗
PubMed1984

Translational regulation and deadenylation of a protamine mRNA during spermiogenesis in the mouse.

The distribution of the mRNA for one of the two mouse protamines, the cysteine-rich, tyrosine-containing protamine (MP1), was examined in the polysomal and nonpolysomal compartments of total testis and purified populations of round and elongating spermatids using Northern blots. In postmitochondrial supernatants prepared from total testis, about 10-15% of MP1-mRNA sediments with the small polysomes. The nonpolysomal molecules of MP1-mRNA are homogeneous in size, about 580 bases, while the polysomal molecules are heterogeneous with a mode of about 450 bases. Digestion with RNase H and thermal chromatography on poly(U) Sepharose reveals that the difference in size of polysomal and nonpolysomal MP1-mRNA is due to a shortening of the poly(A) from about 160 to 30 bases. In round spermatids, essentially all of MP1-mRNA is 580 bases long and is in the nonpolysomal fraction. Elongating spermatids contain roughly equal proportions of the homogeneous, 580 base form in the nonpolysomal compartment, and the heterogeneous 450 base form solely in the polysomal compartment. These results indicate that mRNA for one of the mouse protamines is stored as an untranslated RNP in round spermatids, and that it is partially deadenylated when it is translated in elongating spermatids.

Animals↗
PubMed1984

The differential expression of the actins and tubulins during spermatogenesis in the mouse.

Following intratesticular injection of [35S]methionine, the multiple isoforms of actin and tubulin from highly purified mouse testicular meiotic and post-meiotic cells have been analysed by high resolution two-dimensional gel electrophoresis. In pachytene spermatocytes both beta and gamma actin are synthesized, gamma actin being made in a significantly greater amount. The relative proportion of synthesis of beta and gamma actin changes during spermiogenesis, beta actin increasing and gamma actin decreasing in round spermatids, elongating spermatids, and residual bodies. Both alpha and beta tubulin are synthesized in approximately equal proportion in pachytene spermatocytes. In addition to the tubulin isoforms synthesized during meiosis, at least one new form of both alpha and beta tubulin first appears in post-meiotic (haploid) cells. In elongating spermatids and residual bodies, the synthesis of alpha tubulin is drastically reduced.

Actins↗
PubMed1983

cDNA clones encoding cytoplasmic poly(A)+ RNAs which first appear at detectable levels in haploid phases of spermatogenesis in the mouse.

We have isolated several cDNA clones encoding cytoplasmic poly(A)+ RNAs which are enriched in postmeiotic (haploid) spermatogenic cells in the mouse. Seventeen of 750 clones from a testis cDNA library hybridized more strongly to 32P-labeled cDNA copied from cytoplasmic poly(A) RNA of round spermatids than pachytene spermatocytes. Northern gel blots demonstrated that these 17 plasmids hybridized to RNA(s) approximately 0.5 kb (1 clone), 0.7 kb (13 clones), 0.8 kb (1 clone), and 0.9 kb (2 clones). Four plasmids hybridizing to RNAs 0.7 and 0.9 kb were further characterized by Northern blots. The levels of hybridization were about 10-fold greater with RNA from round spermatids, elongating spermatids and residual bodies than from pachytene spermatocytes from adult testis. These plasmids did not hybridize with cytoplasmic poly(A)+ RNA from sexually immature testis, adult liver, or brain, larger precursors in adult testis nuclear RNA, total RNA from cultured Sertoli cells, poly(A)- RNA from adult testis or the mouse mitochondrial genome. These results demonstrate that certain poly(A)+ RNAs are abundant in haploid cells but barely or not detectable in meiotic cells suggesting the accumulation of these RNAs in round spermatids requires transcription in haploid cells.

Animals↗