Creatine kinase in trout male germ cells: purification, gene expression, and localization in the testis.

High creatine kinase (CK) activity (16.5 +/- 7.6 IU/mg) is present in trout spermatozoa. In order to partly characterize the CK isozyme predominantly present in sperm and to study the expression of this protein in spermatogenesis, we purified to homogeneity a CK (s-CK) from trout sperm, by nitrogen cavitation followed by two chromatography steps (DEAE-Trisacryl and Blue Sepharose). Specific antisera to 5-CK were developed. A cDNA encoding for a CK named TCK1, and whose transcript shows enhanced testicular expression, was previously isolated from trout testis (Garber et al., 1990: Biochim Biophys Acta 1087:256-258). A CK subunit expressed in vitro by this cDNA cross-reacts with anti-s-CK. A 21-amino-acid residue sequence near the N-terminus of s-CK is identical to the cDNA-derived sequence of TCK1, which is unlike any previously reported CK sequence. Using in situ hybridization, the TCK1 mRNA was detectable in primary and secondary spermatocytes and in early spermatids. Immunohistochemical staining of testis and various organs revealed that s-CK was confined to testis and, in this organ, to late spermatids and spermatozoa. In gill, some cells exhibited a positive signal, but another study rules out the presence of s-CK in this organ (Garber et al., 1990: Biochim Biophys Acta 1087:256-258). These results demonstrate that s-CK/TCK1 is a germ cell-specific protein, the transcription of which starts in meiotic germ cells, while translation starts in late spermatids.

Regulation of protamine gene expression in an in vitro homologous system.

An in vitro transcription system from the trout testis nuclei was developed to study trout protamine gene expression. The protamine promoter contains, among others, two regulatory elements: 1) a cAMP-responsive element or CRE element (TGACGTCA) which is present in position 5' to TATA box, and 2) GC box (CCGCCC) which is present in position 3' to TATA box. The removal of the CRE-binding protein by titration (by the addition of appropriate oligonucleotides to the incubation mixture) resulted in a decrease in transcription of the protamine gene. These results were confirmed by experiments in which the pure CRE-binding factor (TPBP1) was used, as well as by those where a stimulatory effect of cAMP on protamine promoter transcription was observed. On the other hand, addition of oligonucleotides containing the GC-box sequence enhanced the protamine gene transcription indicating that the protein (Sp1 like) which binds to this sequence acts as a repressor of protamine gene expression. These results confirm the previously proposed model which suggested that the GC box played a role in negative regulation of the protamine gene expression. Involvement of some other factors in this process was also discussed.

The evolution of protamine P1 genes in dasyurid marsupials.

We report the complete DNA sequences of the protamine P1 gene and flanking regions for 13 species of the marsupial family Dasyuridae. The structure of the protamine locus is conserved in dasyurids and consists of two exons (of lengths 142-151 and 47 bp) separated by an intron (208-240 bp). A key feature of the dasyurid intron is a 38-40 bp duplication found in all species examined to date. This duplication apparently predates the radiation of modern dasyurid lineages and may be homologous to a similar feature in the marsupial mole (Notoryctes). Sequences from a species of Planigale demonstrate that this genus is unique among marsupials in possessing cysteine residues in its protamine P1 molecules. Cysteines may provide enhanced chemical stability for condensed sperm nuclei, a physiological feature that would converge on the common eutherian pattern. Phylogenetic analysis of the protamine genes yields a tree that is largely congruent with previous molecular systematic studies in two areas: (1) There are three main dasyurid lineages corresponding to the Sminthopsinae. Dasyurinae, and Phascogalinae; (2) Dasyurinae and Phascogalinae are sister groups. This study is the first estimate of dasyurid relationships based on a nuclear DNA sequence.

Convergent evolution of cysteine residues in sperm protamines of one genus of marsupials, the Planigales.

A characteristic feature of the sperm P1 protamines of eutherian mammals is the constant presence of six to nine cysteine residues per molecule. During spermiogenesis these residues become oxidized to form a three-dimensional network of disulfide bridges between, and within, protamine molecules in the sperm chromatin. This covalent cross linking strongly stabilizes eutherian sperm nuclei. In contrast, protamines sequenced from teleost fish, birds, monotremes, and marsupials all lack cysteine residues and their sperm nuclei, without the stabilizing cross links, are easily decondensed in vitro. We have now found that one genus of tiny, shrewlike dasyurid marsupials, the Planigales, possess P1 protamines containing five to six cysteine residues. These residues appear to have evolved since the divergence of Planigales from other members of the family Dasyuridae, such as the marsupial mouse, Sminthopsis crassicaudata. We believe this constitutes a case of convergent evolution in a subfamily of dasyurid marsupials toward the cysteine-rich eutherian form of sperm protamine P1.

cDNA sequence and structure of a trout HMG-2 gene. Evidence for a trout-specific 3'-untranslated region.

A complete cDNA sequence (1026 bp) and a partial structure of a gene encoding the trout testis chromosomal HMG (high mobility group) 2 protein (HMG-T2) is presented. The deduced protein consists of 214 amino acids and shares over 80% similarity to the trout HMG-1 protein (HMG-T1) as well as to the mammalian or avian HMG-2 proteins. Northern blot analysis revealed two transcripts, a major one of 1.2 kb and a minor one of 1.6 kb. Southern analysis and polymerase chain reaction of trout genomic DNA indicated that the HMG-T2 gene is encoded by several introncontaining genes. The 5'-UTR (untranslated region) of the HMG-T2 is interrupted by an intron and the coding region of the HMG-T2 is divided into four exons by three relatively short introns (173, 91 and 78 bp). The exon/intron boundaries of trout HMG-2 are identical to those of human HMG-2, as reported earlier [Shirakawa and Yoshida, J. Biol. Chem. 267 (1992) 6641-6645], suggesting the evolution of the HMG-1/2 family genes from a common ancestor. Phylogenetic analysis indicated that the common ancestor of trout HMG-1/2 genes very likely diverged from the ancestor of the mammalian (or avian) HMG-1/2 genes before its separation into two distinct mammalian or (avian) HMG-1 and HMG-2 genes. Sequence comparisons of the 3'UTR of trout HMG-2 cDNA with the corresponding regions in the mammalian (or avian) HMG-2 revealed that the trout 3'-UTR was clearly distinct from the 3'-UTR of the mammalian or avian HMG-2 cDNAs which were otherwise remarkably well conserved.(ABSTRACT TRUNCATED AT 250 WORDS)

Evolution of protamine P1 genes in mammals.

Protamine P1 genes have been sequenced following PCR amplification from 11 mammals representing five major mammalian orders: Rodentia (rat and guinea pig), Carnivora (cat and bear), Proboscidea (elephant), Perissodactyla (horse), and Artiodactyla (camel, deer, elk, moose, and gazelle). The predicted amino acid sequence for these genes together with previously reported sequences results in a data set of 25 different P1 genes and 30 different P1 amino acid sequences. The alignment of all these sequences reveals that protamines are amongst the most rapidly diverging proteins studied. In spite of the large number of differences there are conserved motifs that are also common to birds such as the N-terminal ARYR followed by the triple alternating SRSRSR phosphorylation site. The central region contains 3 arginine clusters consisting of 5-6 arginines each. The C-terminus appears to be the most variable region of the protamines. Overall the molecular evolution of P1 genes is in agreement with the expected species evolution supporting that these genes have evolved vertically.

Expression of an avian protamine in transgenic mice disrupts chromatin structure in spermatozoa.

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.

Molecular phylogeny and evolution of marsupial protamine P1 genes.

We report the DNA sequences of protamine P1 genes and flanking regions from 21 mammalian species, including representatives of all extant marsupial orders. The protamine P1 locus in marsupials displays highly conserved 5' and 3' flanking sequences, as well as a highly variable intron. Marsupial protamines examined are distinct from those of eutherian mammals in lacking cysteine residues, a feature that may be correlated with lower stability of marsupial sperm nuclei during chromatin condensation. Phylogenetic analysis of protamine sequences leads to the following conclusions: (i) the microbiothere Dromiciops is part of a clade that includes dasyurids, Notoryctes and diprotodontians but not bandicoots; (ii) dasyurids are the closest living relatives of Notoryctes; (iii) macropodids, phalangerids and pseudocheirids form a clade apart from the phascolarctids; and (iv) the closest living relatives of caenolestids are didelphids.

Antibodies to high mobility group proteins in systemic sclerosis.

OBJECTIVE: To determine the prevalence of autoantibodies to high mobility group (HMG) proteins in systemic sclerosis (SSc). METHODS: One hundred ninety-seven unselected sera from patients diagnosed as SSc (n = 180) or Raynaud's phenomenon (RP) (n = 17) were tested for HMG autoantibodies by ELISA and immunoblotting. RESULTS: Seventy-one of the 180 (39.0%) SSc sera bound to HMG proteins in an ELISA: 56 (31%) to HMG-1 and/or HMG-2; 29 (16%) to HMG-14/17. In the same assay, 7 of 17 RP sera (41%) bound to HMG proteins: 4 (23%) to HMG-1 and/or HMG-2, and 5 (29%) to HMG-14/17. The specificity of HMG binding was confirmed by immunoblotting. CONCLUSION: Antibodies to HMG proteins, particularly to HMG-1 and HMG-2 are found in about 1/3 of SSc Sera. Since HMG-1 and HMG-2 have a role in transcription, these observations further implicate transcriptional complexes as targets of autoantibodies in scleroderma. This is the first published report of HMG autoantibodies in scleroderma.

cDNA sequence and structure of a gene encoding trout testis high-mobility-group-1 protein.

Perchloric acid extraction of trout testis nuclei revealed the presence of two large high-mobility-group (HMG) proteins, HMG-T1 and HMG-T2. The sequence of a complete cDNA (1407 bp) for trout testis HMG-1 protein (referred as to HMG-T1) has been determined. The deduced HMG-T1 protein contains 203 amino acids with more than 86% similarity to mammalian HMG-1 proteins. A single-sized mRNA for HMG-T1 has been detected by Northern-blot analysis consistent with the size derived from the HMG-T1 cDNA. Amplification of human and trout genomic DNAs by polymerase chain reaction using primers specific for trout and human HMG-1 cDNAs revealed that unlike the human genome, which contains predominantly intronless HMG-1 sequences, intronless HMG-T1 sequences were not found in the fish genome. Southern-blot analysis suggested that the trout testis HMG-1 gene is encoded by at least two sequences with high similarity. A gene encoding HMG-T1 protein has been isolated from a trout testis genomic library and by PCR of trout genomic DNA (3879 bp). The trout testis HMG-1 gene is organized into five exons (four exons corresponding to the protein-coding region) and its exon/intron boundaries are identical to those of the human HMG-2 gene [Shirakawa, H. & Yoshida, M. (1992). J. Biol. Chem. 267, 6641-6645] suggesting the evolution of HMG-1 and HMG-2 genes from a common ancestor.

Antibodies to HMG proteins in patients with drug-induced autoimmunity.

OBJECTIVE: To determine the prevalence of autoantibodies to high-mobility group (HMG) proteins in sera from patients with drug-induced lupus (DIL). METHODS: Forty-two patients who developed autoantibodies and/or lupus after treatment with procainamide or other drugs were tested for HMG autoantibodies by immunoblotting and enzyme-linked immunosorbent assay (ELISA). RESULTS: Twenty-eight of the 42 sera (67%) bound HMG-14 and/or HMG-17. In comparison, 9 of 42 (21%) bound HMG-1 and/or HMG-2. There was a good correlation between ELISA results and binding on immunoblots. CONCLUSION: The high prevalence of antibodies to the nucleosomal core HMGs (HMG-14 and HMG-17) in DIL patients adds evidence implicating nucleosomes as immunogens in drug-induced autoimmunity.

Evolution of the monotremes. The sequences of the protamine P1 genes of platypus and echidna.

The protamine P1 genes from two monotremes, platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus) were isolated after polymerase-chain-reaction amplification then cloned and sequenced. The two protamine P1 genes are of 290 bp and 311 bp for platypus and echidna, respectively, and are clearly orthologous to the published sequences of protamine P1 genes of eutherian mammals and birds. Both genes contain an intron, like the mammals and marsupials and unlike the bird P1 genes that are intronless. The deduced protein sequences from the coding areas of the platypus and echidna protamine P1 genes do not contain any cysteine residues. This absence of cysteine residues leaves the sperm nuclei susceptible to disruption in vitro by exposure to increasing ionic strength and is a characteristic of fish, birds and marsupials. In contrast, the P1 protamines of placental mammals invariably contain 6-9 cysteine residues that, as a result of the formation of intermolecular and intramolecular disulfide bridges, significantly increase the stability of the sperm nuclei that can only be disrupted following disulfide-bond cleavage. Phylogenetic analysis of the protamine P1 gene sequences indicates that the monotremes occupy a position half-way between the eutherian mammals and birds. From the DNA sequences we estimate the time of divergence of the platypus and the echidna to be around 22 million years ago. This date agrees very well with the published estimates of divergence based on other criteria.

Evolution of protamine P1 genes in primates.

Protamine P1 genes have been sequenced by PCR amplification and direct DNA sequencing from 9 primates representing 5 major families, Cebidae (new world monkeys), Cercopithecidae (old world monkeys), Hylobatidae (gibbons), Pongidae (gorilla, orangutan, and chimpanzee), and Hominidae (human). In this recently diverged group of primates these genes are clearly orthologous but very variable, both at the DNA level and in their expressed amino acid sequences. The rate of variation amongst the protamine P1s indicates that they are amongst the most rapidly diverging polypeptides studied. However, some regions are conserved both in primates and generally in other placental mammals. These are the 13 N-terminal residues (including a region of alternating serine and arginine residues (the motif SRSR, res. 10-13) susceptible to Ser phosphorylation), a tract of six Arg residues (res. 24-29) in the center of the molecule, and a six-residue region (RCCRRR, res. 39-44), consisting of a pair of cysteines flanked by arginines. Detailed consideration of nearest-neighbor matrices and trees based on maximum parsimony indicates that P1 genes from humans, gorillas, and chimpanzees are very similar. The amino acid and nucleotide differences between humans and gorillas are fewer than those between humans and chimpanzees. This finding is at variance with data from DNA-DNA hybridization and extensive globin and mitochondrial DNA sequences which place human and chimpanzee as closest relatives in the super family, Hominoidea. This may be related to the fact that protamine P1s are expressed in germ line rather than somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a marsupial sperm protamine gene and its transcripts from the North American opossum (Didelphis marsupialis).

A synthetic oligonucleotide primer, designed from marsupial protamine protein-sequence data [Balhorn, R., Corzett, M., Matrimas, J. A., Cummins, J. & Faden, B. (1989) Analysis of protamines isolated from two marsupials, the ring-tailed wallaby and gray short-tailed opossum, J. Cell. Biol. 107] was used to amplify, via the polymerase chain reaction, protamine sequences from a North American opossum (Didelphis marsupialis) cDNA. Using the amplified sequences as probes, several protamine cDNA clones were isolated. The protein sequence, predicted from the cDNA sequences, consisted of 57 amino acids, contained a large number of arginine residues and exhibited the sequence ARYR at its amino terminus, which is conserved in avian and most eutherian mammal protamines. Like the true protamines of trout and chicken, the opossum protamine lacked cysteine residues, distinguishing it from placental mammalian protamine 1 (P1 or stable) protamines. Examination of the protamine gene, isolated by polymerase-chain-reaction amplification of genomic DNA, revealed the presence of an intron dividing the protamine-coding region, a common characteristic of all mammalian P1 genes. In addition, extensive sequence identity in the 5' and 3' flanking regions between mouse and opossum sequences classify the marsupial protamine as being closely related to placental mammal P1. Protamine transcripts, in both birds and mammals, are present in two size classes, differing by the length of their poly(A) tails (either short or long). Examination of opossum protamine transcripts by Northern hybridization revealed four distinct mRNA species in the total RNA fraction, two of which were enriched in the poly(A)-rich fraction. Northern-blot analysis, using an intron-specific probe, revealed the presence of intron sequences in two of the four protamine transcripts. If expressed, the corresponding protein from intron-containing transcripts would differ from spliced transcripts by length (49 versus 57 amino acids) and would contain a cysteine residue.

Two salmon neuropeptides encoded by one brain cDNA are structurally related to members of the glucagon superfamily.

A cDNA that codes for two peptides in the glucagon superfamily has been isolated from sockeye salmon brain. The first peptide is related to growth hormone-releasing hormone (GHRH), which has high sequence similarity with PACAP-related peptide. The second peptide is structurally related to vasoactive intestinal peptide, which is also related to a newly identified peptide in mannals, pituitary adenylate-cyclase-activating polypeptide (PACAP). The salmon precursor contains 173 amino acids and has dibasic and monobasic enzyme-processing sites for cleavage of a 45-amino-acid GHRH-like peptide with a free C-terminus and a 38-amino-acid PACAP with an amidated C-terminus. The salmon GHRH-like peptide has 40% amino acid sequence identity with a human GHRH and 56% identity with human PACAP-related peptide. The 38-amino-acid salmon PACAP is highly conserved (89-92% identity) with only three or four amino acid substitutions compared with the human, ovine and rat 38-amino-acid PACAP. Not previously reported for mammalian species, a short precursor coding for only one peptide exists in salmon in addition to the long precursor coding for two peptides. In the short precursor, the coding region for GHRH is deleted leaving the PACAP-coding region in a correct reading frame. This provides one possible control mechanism for an increased expression of one peptide (PACAP) without the concomitant increase in the other peptide (GHRH) as occurs in a double-peptide precursor. The importance of the 3' non-translated region of the salmon GHRH/PACAP precursor in the regulation of translation is suggested by its 70% nucleotide sequence identity to the 3' non-translated regions of the mammalian PACAP precursors. The structural organization of the salmon GHRH/PACAP precursor provides a possible evolutionary scheme for precursors that contain tandem peptides in the glucagon superfamily.

Evolution of pro-protamine P2 genes in primates.

Protamines P1 and P2 form a family of small basic peptides that represent the major sperm proteins in placental mammals. In human and mouse protamine P2 is one of the most abundant sperm proteins. The protamine P2 gene codes for a P2 precursor, pro-P2 which is later processed by proteolytic cleavages in its N-terminal region to form the mature P2 protamines. We have used polymerase chain amplification to directly sequence the pro-P2 genes of the five major primate families: red howler (Alouatta seniculus) is a New World monkey (Cebidae); the two macaque species, Macaca mulatta and M. nemistrina are Old World monkeys (Cercopithecidae), the gibbon, Hylobates lar, represents one branch of the apes (Hylobatidae); the orangutan, Pongo pygmaeus, gorilla, Gorilla gorilla and two species of chimpanzee Pan paniscus and Pan troglodytes represent a second ape family (Pongidae). These pro-P2 genes are compared with that of human [Domenjoud, L., Nussbaum, G., Adham, I. M., Greeske, G. & Engel, W. (1990) Genomics 8, 127-133]. The overall size and organization of the genes are conserved within the group. The mean length of pro-P2 is 101 residues, with an increase to 102 in M. nemistrina and a decrease to 99 residues in red howler (A. seniculus). In gorilla and red howler one of two 79-bp tandem repeats that occurs 3' of the gene is deleted. Of the 101 deduced amino acids examined, an amino acid change occurs in one or more primates at 45 positions. Considering only the most recently diverged group, the human/gorilla/chimpanzee clade, this represents a very high mutation rate of 0.99 changes/100 sites in 10(6) years. This rapid mutation rate is characteristic of both members of the protamine gene family, P1 and P2. Consideration of the variable nature of the sequences at the multiple sites of proteolysis during the processing of the pro-P2 indicates either that there are several processing enzymes of differing specificities, or more likely that the folded structure of the pro-P2 limits accessibility of a non-specific protease to certain exposed sites.

A retropseudogene for non-histone chromosomal protein HMG-1.

Southern analysis of the human genome revealed that there are several sequences with homology to the nonhistone chromosomal protein HMG-1. The majority of the HMG-1 sequences are intronless as suggested from the polymerase chain reaction of HeLa DNA. Sequencing of a clone from a human placenta genomic library revealed that the clone was intronless and displayed 99% homology to the human HMG-1 cDNA. The 5' regulatory motif, CCAAT, is present in the clone but there is no TATA-box. Most of the differences between the HMG-1 cDNA sequence and the clone involve point mutations with no interruption of the reading frame. The sequence is flanked at 5' and 3' ends by a 15 nucleotide long direct repeat suggesting that the clone is a processed HMG-1 retropseudogene. Sequence differences between the reading frames of the HMG-1 pseudogene and HMG-1 cDNA indicated that the pseudogene arose relatively late in evolution, approximately one million years ago. The present paper is the first study on a genomic sequence related to HMG-1 genes.

Analysis of the core histone gene cluster of the annelid Platynereis dumerilii.

The arrangement of the polychaete annelid Platynereis dumerilii core histone gene cluster and nucleotide sequence has been reported (D. Sellos, S. A. Krawetz and G. H. Dixon, 1990). The H2B and H3 mRNAs are transcribed from one DNA strand, while the H2A and H4 histone mRNAs are transcribed from the other. The H1 gene is not contained as a member of this cluster. Computer assisted sequence analysis of this region was undertaken to define the organization and representation of the various sequence motifs embedded within this region. The analysis revealed that two large regions on opposite strands of the cluster were similar to one another. This organization is reminiscent of an ancient gene duplication event from which the various members independently evolved.