Testis Nuclear Extract Research Articles

Deoxyribonucleic acid-protein interactions associated with transcriptional initiation of the mouse testis-specific cytochrome c gene.

Transcriptional regulation of the mouse testis-specific cytochrome c (cyt. cT) gene was studied by examining DNA-protein interactions in its proximal promoter. Testicular and liver nuclear proteins bound to the cyt. cT gene at sites -105 to -81, +87 to +113, and +146 to +169, suggesting interactions with ubiquitous nuclear proteins. Protein present in liver nuclear extracts bound to a fourth site at -176 to -125, whereas protein present in testicular nuclear extracts bound to a subregion of this site at -176 to -140. The sequence from -136 to -127, bound by liver but not testicular nuclear proteins, is similar to that of the binding site of a somatic c-mos repressor protein. Lastly, different nuclear proteins from mouse liver and testis bound to a region from -18 to +31 that contains a putative Y box at -13 to -2. Mobility shift assays, Southwestern blots, and immunoprecipitation studies have established that this putative Y box binds a 52-kDa mouse testicular homologue of the Xenopus germ cell-specific Y-box protein and a competing 50-kDa protein present in both liver and testis nuclear extracts. These data suggest that the testis-specific expression of the mouse cyt. cT gene during spermatogenesis may be regulated by the differential binding of tissue-specific nuclear proteins to its proximal promoter region.

Specific Interaction of DNA Polymerase β and DNA Ligase I in a Multiprotein Base Excision Repair Complex from Bovine Testis

Base excision repair (BER) is a cellular defense mechanism repairing modified bases in DNA. Recently, a G:U repair reaction has been reconstituted with several purified enzymes from Escherichia coli (Dianov, G., and Lindahl, T.(1994) Curr. Biol. 4, 1069-1076). Using bovine testis crude nuclear extract, we have shown that G:U is repaired efficiently in vitro, and DNA polymerase beta (beta-pol) is responsible for the single nucleotide gap-filling synthesis (Singhal, R. K., Prasad, R., and Wilson, S. H.(1995) J. Biol. Chem. 270, 949-957). To investigate potential interaction of beta-pol with other BER protein(s), we developed affinity chromatography matrices by cross-linking purified rat beta-pol or antibody against beta-pol to solid supports. Crude nuclear extract from bovine testis was applied to these affinity columns, which were then extensively washed. Proteins that bound specifically to the affinity columns were co-eluted in a complex with beta-pol. This complex had a molecular mass of approximately 180 kDa and was able to conduct the complete uracil-initiated BER reaction. The BER complex contained both beta-pol and DNA ligase I. An antibody to beta-pol was able to shift the complex in sucrose gradients to a much larger molecular mass (>300 kDa) that again contained both beta-pol and DNA ligase I. Furthermore, DNA ligase I and beta-pol were co-immunoprecipitated from the testis nuclear extract with anti beta-pol IgG. Thus, we conclude that beta-pol and DNA ligase I are components of a multiprotein complex that performs BER.

Partial purification and characterization of two types of homologous DNA pairing activity from rat testis nuclei

We describe the partial purification and characterization of two different types of homologous DNA pairing activity from rat testis nuclear extracts. The activities are separated from each other by single-stranded DNA-cellulose affinity chromatography. One activity requires single-stranded DNA ends and promotes the homologous pairing of single-stranded DNA fragments with double-stranded circular DNA and has an apparent molecular mass of 100 kDa as determined by gel filtration chromatography. This pairing activity does not require the addition of exogenous ATP and is strongly Mg 2+-dependent. The second pairing activity promotes strand-transfer between single-stranded circular DNA and homologous double-stranded DNA fragments and has an apparent molecular mass of 30 kDa as determined by gel filtration chromatography. This pairing activity also does not require ATP but, in contrast to the former, is Mg 2+-independent.

A dual-function palindromic sequence regulates testis-specific transcription of the mouse lactate dehydrogenase c gene in vitro.

The promoter of the mouse lactate dehydrogenase c (Ldh-c) gene was assayed in an in vitro transcription system. Of the nuclear extracts isolated from several mouse tissues, only testis supported transcription from a 710 Ldh-c promoter fragment. Mutation analysis indicated that a canonical TATA box and a 30-bp palindromic sequence are both required for promoter activity. Liver nuclear extract (LN) can reduce transcriptional activity significantly when added to testis nuclear extract (TN). Deletion of the 3' palindromic sequence resulted in a low level of transcriptional activity in LN. Competition with a double-stranded synthetic palindromic oligonucleotide inhibited transcription in TN and rescued a low level of transcription in LN. Our results strongly indicate that the palindromic sequence contains both a negative element for repression in somatic tissues and a positive element for activation of the Ldh-c gene in testis.

A mouse homologue of the Xenopus germ cell-specific ribonucleic acid/deoxyribonucleic acid-binding proteins p54/p56 interacts with the protamine 2 promoter.

Recent evidence indicates that a member of the Y box-binding family of transcriptional regulators is identical to p56, a predominant protein of messenger ribonucleoprotein complexes. The p56 protein is highly enriched in oocytes and testis, and a functional RNA binding mouse cytoplasmic homologue has been cloned and partially characterized. Because few potential testis-specific transcriptional regulators have been identified, the testis-enriched Y box-binding proteins represent trans-acting elements of a unique model system for the study of haploid gene expression. The 5' flanking region of the testis-specific, haploid-expressed mouse protamine 2 gene contains an element with a 9-of-12 nucleotide identity with the previously defined Y box consensus sequence. We have investigated the possible role of Y box-binding proteins in transcriptional regulation of protamine 2 using specific antibodies and DNA-protein binding assays. Western blot analyses with two different anti-p54/p56 antibodies demonstrate that a mouse homologue of Xenopus p54/p56 is present in transcriptionally active mouse testis nuclear extracts. Our results further indicate that the Xenopus Y box-binding proteins bind to an element 5' to the mouse protamine 2 gene. Similarly, binding of the mouse testis homologue to the protamine 2 Y box element is demonstrated by gel mobility shift and antibody supershift analyses. The demonstrated interactions between testis-enriched Y box-binding proteins and protamine 2 transcriptional control elements therefore represent a unique system for functional studies to determine the mechanism of regulation of haploid gene expression.

Identification of two positive transcriptional elements within the 91‐base pair promoter for mouse testis angiotensin converting enzyme (testis ACE)

Testis angiotensin-converting enzyme (testis ACE) is an isozyme of ACE only expressed by male germ cells during spermiogenesis. It is the result of a strong sperm-specific promoter found within the 12th intron of the somatic ACE gene. Previous studies have localized the boundaries of the mouse testis ACE promoter as being from -91 to -9, relative to the transcriptional start site, and have suggested two important DNA regulatory elements starting at positions -55 and -32. DNA constructs were made in which these motifs were either eliminated or substituted. Each construct was tested for its ability to promote transcription in vitro, using a rat testis nuclear extract. Disruption of either motif reduced in vitro transcription to about 30% of control levels, while mutations of both elements abolished transcription. Two sites were selected inside each motif and altered by point mutation. Each of four constructs, containing a mutation at -51, -48, -30, or -28, transcribed at 29% or less the efficiency of the parent construct. The DNA element at -55, TGAGGTCA, is homologous to a consensus cyclic AMP response element. The motif at -32, TCTTAT, is located at a position analogous to a TATA box. Substitution of the -32 motif with a consensus TATA box sequence, TATAAA, stimulated transcriptional activity about 3-fold. As measured by gel mobility shift, oligonucleotides encompassing the -32 motif and the consensus TATA box formed different DNA-protein complexes. However, the -32 motif oligonucleotide was recognized by nuclear proteins prepared from either liver or testis nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Polymerase β Conducts the Gap-filling Step in Uracil-initiated Base Excision Repair in a Bovine Testis Nuclear Extract

The G:U mismatch in genomic DNA mainly arises from deamination of cytosine residues and is repaired by the base excision repair pathway. We found that a bovine testis crude nuclear extract conducts uracil-initiated base excision repair in vitro. A 51-base pair synthetic DNA substrate containing a single G:U mismatch was used, and incorporation of dCMP during repair was exclusively to replace uracil. A neutralizing polyclonal antibody against DNA polymerase beta (beta-pol) inhibited the repair reaction. ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of beta-pol was required. Next, the base excision repair system was reconstituted using partially purified components. Several of the enzymatic activities required were resolved, such that DNA ligase and the uracil-DNA glycosylase/apurinic/apyrimidinic endonuclease activities were separated from the DNA polymerase requirement. We found that purified beta-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases alpha, delta, and epsilon could not. These results with purified proteins corroborated results obtained with the crude extract and indicate that beta-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system.

Calspermin gene transcription is regulated by two cyclic AMP response elements contained in an alternative promoter in the calmodulin kinase IV gene.

The transcript for the high-affinity Ca2+/calmodulin-binding protein calspermin is generated from the gene encoding Ca2+/calmodulin-dependent protein kinase IV only in postmeiotic germ cells during spermatogenesis. We demonstrate that this testis-specific calspermin transcript can be produced in heterologous cells by utilization of a promoter located in an intron of the calmodulin (CaM) kinase IV gene. Critical motifs within this promoter are two cyclic AMP response element (CRE)-like sequences located about -70 and -50 bp upstream of the transcriptional initiation site. Both CRE motifs are footprinted by the authentic testis-specific transcriptional activator CREM tau or by CREM tau present in adult testis nuclear extract. Whereas a 2.1-kb DNA fragment containing the calspermin promoter is inactive when transfected into NIH 3T3 cells, activity can be restored by cotransfection of CREM tau and protein kinase A or CaM kinase IV but not CaM kinase II alpha. Restoration of activity is greatly reduced by mutation of the two CRE motifs. Since CRE-like motifs have been identified in many genes uniquely expressed in postmeiotic germ cells, which contain abundant CREM tau protein, we suggest that CREM tau may function as one transcription factor responsible for the expression of postmeiotic germ cell-specific genes.

Identification of a functional cyclic adenosine 3',5'-monophosphate response element in the 5'-flanking region of the gene for transition protein 1 (TP1), a basic chromosomal protein of mammalian spermatids.

Transition protein 1 (TP1) is a small basic chromosomal protein that appears in mammalian spermatids during the period of chromatin condensation. The gene for TP1 from several species contains an apparent cAMP response element (CRE) in the immediate 5'-flanking region. The recent identification of high expression of the novel CRE-activating protein (CREM tau) in advanced testicular germ cells provided a stimulus to ask whether or not the TP1 CRE is functional. To this end we show both by gel retardation and by footprint assays that TP1 CRE forms specific bound complexes with proteins in whole testis nuclear extracts and that these complexes involve CREM as evidenced by recognition by a specific antibody. In addition, the TP1 CRE forms specific bound complexes with bacterially expressed CREM tau. Finally, the TPI CRE conveys protein kinase A-dependent induction to a linked chloramphenicol acetyl transferase gene when transfected into JEG-3 cells. Accordingly, TP1 is a good candidate for a testis-specific gene subject to CREM tau regulation.

A 60-bp core promoter sequence of murine lactate dehydrogenase C is sufficient to direct testis-specific transcription in vitro.

A clone containing the 5' flanking region of the testis-specific murine lactate dehydrogenase C (Ldhc) gene was isolated from a mouse genomic library. Promoter activity was demonstrated within a 720-bp fragment in testis nuclear extract (TN). Interestingly, the addition of liver nuclear extract (LN) significantly repressed Ldhc promoter activity in the transcription assay system. Sequence analysis of this promoter region revealed several ubiquitous cis-regulatory elements, including one TATA box, one GC box, and two putative CCAAT elements. Analysis of a series of deletion mutants revealed that a 60-bp core promoter sequence was sufficient to direct basal, testis-specific transcription in an in vitro transcription system. A 103-kDa protein in TN and a 65-kDa protein in LN bind to the same palindromic sequences within the 60-bp core promoter region.

Testis-specific transcription start site in the aspartate aminotransferase housekeeping gene promoter.

We have studied the expression and regulation of the rat testis cytosolic aspartate aminotransferase gene. The cytosolic aspartate aminotransferase activity was 5-fold lower in the testis than in the liver and kidney. A 1.9-kilobase mRNA form was detected in the rat testis in contrast to the 2.1- and 1.8-kilobase forms present in other organs. Using Northern blot and S1 mapping analyses, we found that the proximal polyadenylation site was almost exclusively used in the testis as opposed to other organs where the distal site was preferentially used. RNase protection and primer extension analysis showed that transcription was initiated at multiple sites in all organs, but the pattern of those start sites was different in the testis; in particular, a novel transcription start site was specifically detected in this organ (at position -115 from the translation start site). This site was first observed in 29-day-old rats and was maximally utilized in the adult testis. DNase I footprinting using testis nuclear extracts revealed the presence of three sites of DNA-protein interaction in the 250-base pair proximal promoter, a pattern similar to the one found using liver nuclear extracts. However, the proteins bound had different properties as shown by gel retardation experiments. We conclude that the pattern of transcription initiation and the polyadenylation site selection of a housekeeping gene can be tissue-specific.

Demonstration of a testis-specific trans-acting factor Tet-1 in vitro that binds to the promoter of the mouse protamine 1 gene.

We have established testis-specific in vitro transcription of the mouse protamine 1 (MP1) gene using rat testis nuclear extracts. Addition of testis nuclear extracts to brain extracts enhanced transcription from the MP1 upstream sequence-carrying adenovirus major late promoter. Moreover, the MP1 upstream region from positions -92 to -41 alone exhibited transcriptional activation in a tissue-specific manner. DNase I footprinting demonstrated the presence of a DNA-binding factor around position -60 (Tet-1) in testis nuclear extracts, but not in other tissues. Gel shift analysis also revealed the presence of testis-specific Tet-1. Since mutational analysis in transcriptional and binding assays demonstrates that the Tet-1 site is responsible for transcriptional activation, we suggest that Tet-1 is a novel tissue-specific trans-acting factor. The Tet-1-recognizing sequence was delineated to the 11-mer TGACTTCATAA at position -64. Although the first 8-mer in the Tet-1 11-mer shares homology with the cyclic AMP-responsive element, Tet-1 is demonstrated to be distinct from known cAMP-responsive element-binding factors.

Characterization of Interaction between Nuclear T3Receptors and Antiserum against Cellular-erb A Peptide*

In our previous study, we raised a polyclonal antiserum against a synthetic 15 amino acid peptide encoding the putative hormone binding domain of c-erb A, which not only inhibited T3-binding to rat liver nuclear T3 receptor (NT3R) but also immunoprecipitated [125I]T3-NT3R complex. In the present study, interactions between this antiserum (4 BII-immunoglobulin G (IgG] and NT3R were further characterized. 4BII-IgG interefered T3 from binding to rat liver NT3R both in the nuclear extract and in isolated nuclei in a similar manner. Preincubation with 4BII-IgG decreased the association constant value with no effect on maximal binding capacity of NT3R. Lineweaver-Burk plot revealed competitive inhibition of T3-NT3R binding. Both the inhibition of T3 binding and immunoprecipitation of NT3R by the antiserum were similarly observed with rat liver, kidney, brain, and testis, and human kidney nuclear extract. Since the polypeptide sequence which was used for immunization is highly homologous between c-erb A alpha 1 and beta, it is likely that 4BII-IgG recognizes both c-erb A alpha 1 and beta in various tissues. On the contrary, 4BII-IgG did not interfere with T3 binding to rat liver cytosol T3-binding protein and thyroxine-binding globulin (TBG) prepared from human serum, nor immunoprecipitated these binding proteins labeled with [125I]T3. The binding protein for reverse T3 (NrT3BP) in the rat liver nuclear extract was neither immunoprecipitated nor influenced in its rT3-binding activity by 4BII-IgG. When the immune complex between the nuclear extract and 4BII-IgG was removed by antirabbit IgG goat serum, the remaining T3-binding activity was reduced by 87%, while no rT3-binding activity was immunodepleted, suggesting that NrT3BP is a protein different from NT3R. The data show that the antiserum against c-erb A peptide recognizes NT3R specifically with no distinction between difference in tissues and species. Carboxyl-terminal region of c-erb A/NT3R is critical for T3-binding.