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Biology of Reproduction 64, 507-517 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

TFIIA{alpha}/ß-Like Factor Is Encoded by a Germ Cell-Specific Gene Whose Expression Is Up-Regulated with Other General Transcription Factors During Spermatogenesis in the Mouse1

SangYoon Hana, Liping Zhoub, Ashok Upadhyayaa, Sang Hyun Leea, Keith L. Parkerb, and Jeff DeJong2,a

a The University of Texas at Dallas, Department of Molecular and Cell Biology, Richardson, Texas 75080 b The University of Texas Southwestern Medical Center, Departments of Internal Medicine and Pharmacology, Dallas, Texas 75235

ABSTRACT

TFIIA{alpha}/ß-like factor (ALF) is a testis-specific counterpart of the large subunit of human general transcription factor TFIIA. Northern analysis shows that ALF mRNA first appears in mouse testis at Postnatal Day 14. Similarly, expression of the general transcription factors TBP, TRF2, TFIIA{alpha}/ß, TFIIA{gamma}, and TFIIIB90 is also increased beginning at Postnatal Day 14, suggesting that there is a coordinated induction of many general transcription factors during male germ cell differentiation. Analysis of male germ cells separated by Staput sedimentation shows that ALF is present in pachytene spermatocytes and haploid spermatids. In addition, in situ hybridization experiments with adult mouse testis shows that ALF is present in haploid spermatids. Searches of the human genome sequence database using the basic local alignment search tool reveal that the ALF and TFIIA{alpha}/ß (GTF2A1) genes are both composed of nine exons, whereas the TFIIA{gamma} (GTF2A2) gene is composed of five exons. Furthermore, nucleotide and amino acid comparisons among human and mouse ALF, TFIIA{alpha}/ß, and TFIIA{gamma} cDNA sequences show that ALF has diverged more rapidly than either TFIIA{alpha}/ß or TFIIA{gamma}. Finally, the ALF and SBLF (Stoned B-Like Factor) sequences present in the chimeric SALF cDNA are both present on human chromosome 2, and an analysis of the corresponding genes suggests a model for the formation of SALF.

developmental biology, gametogenesis, gene regulation, meiosis, spermatid, testes

INTRODUCTION

Accurate promoter recognition and initiation of mRNA synthesis require the assembly of a preinitiation complex (PIC) composed of the RNA polymerase II holoenzyme and a set of general transcription factors (GTFs; TFIIA, -B, -D, -E, -F, and -H) at the core promoters of class II genes [1, 2]. Recent studies have revealed several tissue-specific GTFs, raising the idea that PICs with distinct core promoter specificity, activator specificity, or both, may be utilized at some promoters.

The mRNAs for several GTFs and GTF-related factors display unique patterns of expression in testis. For instance, the TFIIA{alpha}/ß-like factor, ALF, is expressed only in this tissue, while TFIIA{alpha}/ß, TFIIA{gamma}, and TFIIB are expressed at higher levels [3–5]. In addition, the TATA-binding protein (TBP) is up-regulated during the early pachytene stage of meiosis, and its mRNA is estimated to be up to 1000-fold higher in haploid spermatids than in other cell types [3, 6, 7]. A TBP-related factor, TRF1, is preferentially expressed in the gonads and central nervous system (CNS) of adult Drosophila, and mutations in this gene result in male sterility [8, 9]. Another TBP-related factor, TRF2 (also called TLF, TRF, TRP, and TLP), has been identified in human, mouse, chicken, Drosophila, and C. elegans [10–16]. Like TBP, TRF2 transcripts are also present at substantially higher levels in human and mouse testis [13, 15, 16]. Finally, genes for two transcription elongation factors, SII-T1 and Elongin A2, are expressed only in testis [17, 18]. Together, these observations suggest a unique role for the RNA polymerase II transcription machinery in testis, perhaps for germ cell-specific gene regulation.

In this paper we extend our characterization of the ALF gene, focusing on its structure and regulation. Previous studies have shown that the recombinant ALF protein, in conjunction with the small ({gamma}) subunit of TFIIA, can stabilize TBP-DNA interactions and can support basal and activated transcription by RNA polymerase II in vitro [4, 5]. The ALF sequence is also present as part of a less abundant transcript, SALF [4], which contains an upstream sequence from a gene that is similar to the membrane trafficking factor, Stoned B [19], and to the µ-chains of clathrin-associated adaptor complexes [20]. In this report we address three main questions: Do other organisms possess a gene for a testis-specific TFIIA large subunit, and what are the evolutionary relationships among such genes? Is the expression of ALF and other GTFs in testis localized within the germ cells, as is the case for TBP? Finally, what is the molecular basis for the formation of the chimeric human SALF transcript?

MATERIALS AND METHODS

Complementary DNA Cloning

Mouse ALF The 3' end of mouse ALF was isolated in polymerase chain reactions (PCRs) using 4 µl of mouse testis "Marathon" cDNA library (Clontech, Palo Alto, CA) with 10 pmol of primer 2a2–19 (5'-GACTTGAAGCAGCTCTGGGAAACCAAGG-3') and the library-specific adaptor primer, AP-1 (5'-CCATCCTAATACGACTCACTATAGGGC-3'; Clontech). The 1662-base pair (bp) product was subcloned into pGEM-T Easy (Promega, Madison, WI) and sequenced. The 5' end of mouse ALF was isolated in PCR reactions using the MALF-1 (5'-GACTTTGTAAAGGTGACCAGATG-3') and AP-1 primers, followed by reamplification with the nested primers, MALF-2 (5'-GTGGATCGGATAGCCAGCAAA-3') and AP-2 (5'-ACTCACTATAGGGCTCGAGCGGC-3'). The product, which was about 400 bp, was subcloned into pGEM-T Easy (Promega) for sequencing. These overlapping cDNAs were combined to form the composite mouse ALF sequence. Subsequent PCR reactions using EmALF1 (5'-ATTATGCTAGCGCCTTCATCAACCTGGTGCCCA-3') and EmALF2 (5'-TAGACGAGCTCTTACCACTCAGCTTCACCAATG-3') primers that span the open reading frame verified the existence of the full-length transcript.

Mouse TFIIA{alpha}/ß Mouse TFIIA{alpha}/ß was isolated in PCR reactions using 3 µl of mouse testis "Marathon" cDNA library (Clontech) with primers mTFIIAL-1 (5'-ATGTCCCAAGTTGCAAACCGTCC-3') and mTFIIAL-2 (5'-GTGTTGTGTGTGGAAATGGCGAAC-3') based on partial mouse TFIIA{alpha}/ß expressed sequence tags (ESTs; GenBank accession numbers AA536742 and AA119905).

Mouse TFIIA{gamma} A full-length mouse TFIIA{gamma} EST clone (GenBank accession number AA060082) was identified using human TFIIA{gamma} as the query.

Rat TFIIA{alpha}/ß Rat TFIIA{alpha}/ß was cloned using a combination of reverse transcription-PCR (RT-PCR) and library screening. First-strand cDNA was synthesized from poly(A)+ RNA (Clontech) using a random hexamer p(dN)6 (Roche, Indianapolis, IN). This cDNA was used as a template for PCR with degenerate, inosine-containing primers rIIA-1 (5'-GAIGAIGTIATIAAIGAIGTICGIGAIIKITTYYTIGAIGAIGGIGTIGAIGAICA-3') and rIIA-2 (5'-TTCATIATICCRTCYTTIAIITIRAAYTTCCA-3'). The product of this reaction was subcloned into pCR II (Invitrogen, Carlsbad, CA) and used to probe a rat liver 5'-Stretch Plus {lambda}gt11 cDNA library (Clontech). A full-length clone was isolated, subcloned into pBluescript II KS+, and sequenced.

Rat TFIIA{gamma} Rat TFIIA{gamma} was cloned using the insert of pRSEThp12 [21] as a probe to directly screen a rat liver 5'-Stretch Plus {lambda}gt11 cDNA library.

Human TRF2 A sequence related to TBP (GenBank accession number W07871) was identified by a basic local alignment search tool (BLAST) search using human TBP as the query. This clone was obtained from Research Genetics (Huntsville, AL) and sequenced. The clone is 1286 bp long, including a 36 nt poly(A) tail, and encodes a 186-amino acid protein that is about 70% similar to human TBP. The clone is identical to TRF2 [13, 15, 16].

Genomic PCR

Human genomic sequences flanking the junction sites in the ALF and SALF cDNAs were isolated using genomic PCR as described in the GenomeWalker system (Clontech). PCRs were performed with the appropriate combination of adaptor primers (Clontech) AP-1 (5'-GTAATACGACTCACTATAGGGC-3') and AP-2 (5'-ACTATAGGGCACGCGTGGT-3'), ALF-specific primers ALF-1 (5'-GCTGGAGGTGCTGTCATGGC-3') and ALF-4 (5'-GTGCTGTCATGGCCTGCCTCAA-3'), and SBLF-specific primers hSALF-1 (5'-GTGATGTCCAGCCACAGAAACA1-3'), hSALF-2 (5'-GGTCAGGTCTCTGGGAGTGGAG-3'), hSALF-3 (5'-CCTTCAATTACATCTTCAATTACA-3'), and hSALF-4 (5'-AGCAAATAGATTCCGAACTCCTTC-3'). PCRs were typically performed using Elongase (Life Technologies, Grand Island, NY) with 5 ng of genomic library DNA, and products were subcloned into pGEM-T Easy (Promega) for sequencing.

The human chromosomes on which ALF, SBLF, and TFIIA{alpha}/ß sequences are located were determined by genomic PCR of polychromosomal somatic cell hybrid DNAs (Q Biogene, Carlsbad, CA) according to the manufacturer's instructions. Primers hALF-6 (5'-TCCACTCAAACCCGCTCCATCT-3') and hALF-7 (5'-ACAAAAGGTGCAGGGTTCGGCT-3') were used to amplify a 270-bp ALF-specific product. Primers hSALF-1 and hSALF-5 (5'-TCTAGGGCAAATCTCACCACAC-3') were used to amplify a 173-bp SBLF-specific product. Primers hTFIIAL-5 (5'-TGGATTTCATTCAGAAGAGCAGCAGCT-3') and hTFIIAL-8 (5'-GATGCATTCATATGCTGTATCAACTTAGAATCTG-3') were used to amplify a 2.5-kilobase (kb) TFIIA{alpha}/ß-specific product that spans a 2.2-kb intron. DNA templates that produced PCR products were then compared with the chromosome complement of the somatic cell hybrid panel.

Genome Database Search and Analysis

Human ALF gene A search of the high throughput genomic sequence (htgs) database at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/) revealed a bacterial artificial chromosome (BAC) clone from the Washington University Genome Sequencing Center that contains unordered human genomic sequences (chromosome 2) that encodes the ALF gene (GenBank accession number AC073082).

Human SBLF gene A search of the htgs database revealed that the BAC clone (AC073082) also contains a complete Stoned B-Like Factor (SBLF) gene.

Human TFIIA{alpha}/ß gene A search of the htgs database revealed a BAC from the Genoscope Sequencing Center that contains unordered human genomic sequences (chromosome 14) that encodes the TFIIA{alpha}/ß gene (GenBank accession number AL136040).

Human TFIIA{gamma} gene A search of the htgs database revealed a BAC from the Washington University Genome Sequencing Center that contains unordered human genomic sequences (chromosome 15) that encodes the TFIIA{gamma} gene (GenBank accession number AC022480). A putative processed TFIIA{gamma} pseudogene was also identified in sequences from chromosome 8 (GenBank accession number AF252825). This TFIIA{gamma}-like sequence contains an Alu element in the 5'-UTR, and the open reading frame is not continuous.

Northern Analysis

Multiple tissue Northern blots containing polyA+ RNAs from six different tissues of rat and mouse were obtained from Origene. RNAs from rat reproductive tissues (Zivic-Miller, Zelienople, PA), and from the testes of CD-1 mice (Charles River Laboratories, Wilmington, MA) isolated at 6, 10, 12, 14, 16, 18, 20, 22, 24, 27–30, and 40 days after birth were prepared using the method of Chomczynski and Sacchi [22].

Agarose gels (1.5%) for Northern blots were prepared in 1x MEA (20 mM MOPS, 8 mM Na acetate, 1 mM EDTA), and 2.2 M formaldehyde. RNAs (15 µg) were transferred overnight onto Zeta-probe nylon membranes (BioRad, Hercules, CA) and irradiated for 55 sec at 100 mJ/cm2 using an ultraviolet cross-linker (Fisher, Pittsburgh, PA). Filters were prehybridized in 5 ml/cm2 ExpressHyb solution (Clontech) at 68°C for 30 min and hybridized with 10 x 106 cpm/ml of [{alpha}-32P]dCTP-labeled DNA probe at 68°C for 1 h, or with 10 x 106 cpm/ml of [{gamma}-32P]ATP-labeled oligonucleotides. Probes were prepared by random primer labeling of DNA fragments using the Ready-to-Go system (Amersham Pharmacia Biotech, Uppsala, Sweden) or, for oligonucleotides, by T4 kinase labeling with [{gamma}-32P]ATP. Following hybridization, membranes were washed in 2x SSC and 1.0% SDS followed by 0.1x SSC and 0.5% SDS, and were exposed for several hours to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA) or for 1–7 days with XAR-5 film (Kodak, Rochester, NY). For developmental expression studies, several identical blots were prepared so that probes could be used in parallel, and some were stripped and reprobed.

The probes for Northern experiments were as follows: hALF, a 447-bp HincII-HindIII fragment or an 895-bp HincII-BglII fragment; mALF, a 345-bp PstI fragment or a 504-bp PstI-EcoRI fragment; mTFIIA{alpha}/ß, a 315-bp PstI fragment; TFIIA{gamma}, a 355-bp NdeI-BamHI fragment; TRF2, a 607-bp EcoRI-NotI fragment; TFIIIB90, (5'-ATACTTCATGGTTCTTCTCCCCAAACTCCAGCAGATGAGCAAAGCGTGGAATGTA-3'); TBP, a 197-bp PvuII fragment; Protamine-1, (5'-GAAGTCTGGTAAAATTCTCACGCAGGAGTTTTGATGGACTTGCTATTCTGTGCAT-3'); and a partial ß-actin cDNA.

Staput Cell Separation

The EDTA-Trypsin method for the isolation of male germ cells by Staput sedimentation was used with minor modifications [23–25]. Testes from two adult CD-1 mice were minced in 10 ml of PBS using a single razor blade. Cells were filtered through an 80-mesh screen and incubated with 0.25% EDTA-trypsin (Life Technologies) and 20 µg/ml of DNase I (Sigma, St. Louis, MO) for 10 min at room temperature. Cells were then centrifuged at 500 x g for 20 min and resuspended at 2 x 106 cells/ml in PBS containing 20 µg/ml of DNase, 0.002% purified soybean trypsin inhibitor (Sigma), and 0.5% BSA (Fisher). The final volume was between 20 and 30 ml.

Cells were loaded into a 12.5-cm-diameter Staput chamber (Kimble/Kontes, Vineland, NJ) and separated over an 800 ml linear gradient of BSA (1%–4%) for 3-4 h. Fractions (20–40 ml) were collected over a period of 1 h. Aliquots from each fraction were fixed with Bouins fixative, stained with periodic acid-Schiff base, counterstained with hematoxylin, and counted. Fractions 4, 9, and 20 were enriched in pachytene spermatocytes, round spermatids, and elongating spermatids, respectively, and were used to prepare RNA.

Reverse Transcription-Polymerase Chain Reaction

Total RNA from isolated germ cell fractions was analyzed using the Advantage RT-for-PCR kit (Clontech). First-strand cDNA was synthesized from total RNA using MMLV reverse transcriptase at 42°C for 1 h. PCRs (22-30 cycles) were performed with first-strand cDNA that had been normalized for the production of similar levels of a 944-bp ß-actin product using the mActn-11 (5'-AGGACTCCTATGTGGGTGACG-3') and mActn-12 (5'-CTCATCGTACTCCTGCTTGCT-3') primers. Primers for a mouse TFIIA{alpha}/ß product (654 bp) were mTFIIAL-1 (5'-GTGTTGTGTGTGGAAATGGCGAAC-3') and TFIIAL-4 (5'-TCCAGGAAGTGGGGCTAAGA-3'). Primers for a mouse ALF product (904 bp) were mALF-3 (5'-GTTTTACGCCGGAAGACCTGA-3') and mALF-5 (5'-GTCCTCGTTGTCGCTGCTA-3'). Primers for a protamine-1 product (136 bp) were mPrtm-2 (5'-AGATACCGATGCTGCCGCAGC-3') and mPrtm-4 (5'-CACCTTATGGTGTATGAGCGGC-3').

In Situ Hybridization

In situ hybridizations were performed using [{alpha}-35S] uridine 5'-triphosphate (UTP)-labeled antisense and sense RNA probes prepared from human or mouse ALF cDNA constructs. A 447-bp HincII-HindIII fragment from human ALF was subcloned into pBluescript II KS+; the antisense probe was prepared using T7 polymerase and the sense probe was prepared with T3 polymerase. A 345-bp PstI fragment from mouse ALF was subcloned into pBluescript II KS+; the antisense probe was prepared using T3 polymerase and the sense probe was prepared with the T7 polymerase.

Testes of adult mice were perfused with 4% paraformaldehyde, embedded in paraffin, and sectioned at 7 µm. Sections were deparaffinized and hybridized with the labeled sense or antisense transcripts using reagents in the SureSite II kit (Novagen, Milwaukee, WI) according to the recommended protocol. Exposure times were generally between 2 to 4 wk. Counterstaining was with methyl green or hematoxylin/eosin.

RESULTS

Expression of a Testis-Specific ALF Gene in Rodents

To determine whether rodents express ALF we examined RNA from various mouse and rat tissues. Northern analysis revealed a 1.8-kb mRNA species present in mouse testis (Fig. 1A, lane 5), but not in brain, heart, kidney, lung, or skin (lanes 1–4, 6). Likewise, rat testis RNA contained a 1.8-kb mRNA species (Fig. 1B, lane 5) that was not present in other tissues tested (lanes 1–4, 6). We also examined whether ALF was expressed in other male reproductive tissues. The results (Fig. 1B) show that ALF is not present in RNA from rat seminal vesicles (lane 7), vas deferens (lane 8), or epididymis (lane 9). Thus, rodents possess an ALF gene that expresses a testis-specific mRNA similar to that observed in humans. A partial ALF cDNA sequence has also been identified from pig (GenBank accession number AW416121), suggesting that ALF is present in other higher organisms. In contrast, a TFIIA{alpha}/ß-like factor was not identified in database searches of the Saccharomyces cerevisiae, C. elegans, or D. melanogaster genomes (data not shown). It is currently unknown whether TFIIA{alpha}/ß or ALF is orthologous to the single TFIIA large subunit present in these species.



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  		FIG. 1. ALF is selectively expressed in mouse and rat testis. A) Northern blot analysis of RNA from various mouse tissues using an ALF-specific probe reveals a 1.8-kb signal only in testis (lane 5). B) Analysis of ALF expression in various rat tissues shows expression only in testis (lanes 5 and 10). Control experiments using a ß-actin-specific probe show the normalization of RNA on each blot

Cloning of Rodent ALF, TFIIA{alpha}/ß, and TFIIA{gamma} cDNAs

Using a mouse testis cDNA library, we performed PCRs to isolate overlapping cDNAs that encode mouse ALF. The full-length composite sequence is 1614 bp, including a 27-bp poly(A) tail. The 1407-bp open reading frame encodes a 468-amino acid protein with a predicted mass of 51.5 kDa. A rodent counterpart of the chimeric human SALF cDNA was not detected in Northern analysis or in preliminary PCR experiments (data not shown).

We also isolated TFIIA{alpha}/ß subunit cDNAs from both mouse and rat. A mouse TFIIA{alpha}/ß cDNA was isolated from a mouse testis cDNA library using PCR. The full-length, 1227-bp clone predicts a protein of 378 amino acids with a mass of 41.5 kDa. A rat TFIIA{alpha}/ß cDNA was isolated using both RT-PCR and direct hybridization of a rat liver cDNA library. This 1212-bp clone predicts a protein of 377 amino acids with a mass of 41.6 kDa.

Mouse and rat TFIIA{gamma} cDNAs were also isolated. A full-length mouse TFIIA{gamma} was identified in EST database searches using human TFIIA{gamma} as the query. This clone predicts a 109-amino acid protein with a mass of 12.5 kDa. The rat TFIIA{gamma} cDNA was isolated by direct hybridization of a rat liver cDNA library. This 659-bp cDNA encodes a polypeptide identical to that of mouse TFIIA{gamma}. In contrast, human TFIIA{gamma} has a single amino acid variation at position 46 (alanine vs. serine).

Comparison of ALF and TFIIA{alpha}/ß Amino Acid Sequences

An alignment between the amino acid sequence of human ALF with mouse ALF cloned in this report reveals an overall similarity of 77% (Fig. 2A and Table 1). However, the N-terminal 60 amino acids (region I as defined in [29]) and C-terminal 62 residues (region IV) are 92% and 100% similar, respectively (region III is mainly conserved with respect to overall negative charge and is not included in this analysis). The conservation of regions I and IV reflects the importance of these domains for subunit-subunit interactions and TFIIA activity.



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  		FIG. 2. A) The deduced amino acid sequence of a cloned mouse ALF cDNA (M-ALF) is aligned with human ALF (H-ALF). Sequences are numbered to the left of each line. B) The deduced amino acid sequences of cloned mouse and rat TFIIA{alpha}/ß cDNAs (M-{alpha}/ß and R-{alpha}/ß) are aligned with human TFIIA{alpha}/ß (H-{alpha}/ß). C) The deduced amino acid sequences of cloned mouse and rat TFIIA{gamma} cDNAs (M-{gamma} and R-{gamma}) are aligned with human TFIIA{gamma} (H-{gamma}). Shading indicates residues that are similar, while dashes indicate gaps. The dashed, arrowed lines above the alignment denote exon sizes and the positions of exon-intron junctions in the human genes. The open arrows at the ends indicate the continuation into 5'- and 3'-UTR sequences


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  		TABLE 1. Amino acid and nucleotide sequence comparisons between selected human and mouse general transcription factors.*

Region II from human and mouse ALF, defined here as residues 61–339, is only 68% similar. Moreover, of the 100 amino acid substitutions within region II, only 13 were conservative changes, suggesting this sequence is under weak selective pressure. Related, alanine scanning mutations and deletions within region II of the yeast toa1 gene had little effect on TFIIA activity in vitro or in vivo [26]. These observations support the idea that region II is a flexible spacer, and imply that it is the highly conserved TFIIA{gamma} subunit that dictates the proximity and disposition of the N- and C-terminal ends of the large subunit [27, 28]. It is still possible, however, that region II could have a gene- or activator-specific role that may be related to its divergent sequence.

An alignment between the amino acid sequence of human TFIIA{alpha}/ß [29, 30] with the rat and mouse TFIIA{alpha}/ß subunit cDNAs cloned in this report is shown in Figure 2B. The comparison shows an overall similarity of 97%, with regions I and IV between 97–100% similar. In contrast to ALF, region II from human and mouse TFIIA{alpha}/ß (residues 61–273) is also very similar (95%). An alignment between the amino acid sequence of human TFIIA{gamma} [21, 31, 32] with the mouse and rat TFIIA{gamma} subunits reported here is shown in Figure 2C. These sequences are 99% similar (Table 1). Finally, a comparison between human and mouse TBP shows 98% similarity, while human TRF2 is identical to mouse TRF2 (Table 1).

Rapid Evolution of the Human and Mouse ALF Genes

To determine the rate of synonymous substitutions (those that do not result in an amino acid replacement) per synonymous site (KS) and nonsynonymous substitutions (those that do result in an amino acid replacement) per nonsynonymous site (KA) between the human and mouse ALF, TFIIA{alpha}/ß, and TFIIA{gamma} sequences, we used the MEGA program [33]. This analysis is presented in Table 1.

With respect to KS we find the following patterns: 1) Of the three sequence pairs examined (ALF, TFIIA{alpha}/ß, and TFIIA{gamma}), the highest substitution rate was seen with ALF (KS = 0.99 ± 0.10) and the lowest rate with TFIIA{gamma} (KS = 0.24 ± 0.064). 2) There is an apparent decrease in KS within the ALF sequence itself in the order of region I > region II > region IV, with a high value of 1.42 ± 0.51 (region I) and a low value of 0.52 ± 0.16 (region IV). Although the standard deviation in some of these calculations is relatively large due to the short sequence lengths and high degree of divergence, the data indicate that the rate of synonymous substitutions (KS) for ALF is, on average, twofold higher than the rate observed with TFIIA{alpha}/ß.

With respect to KA we find the following patterns: 1) Of the three sequences, the highest substitution rate was again seen with ALF (KA = 0.16 ± 0.013) and the lowest rate with TFIIA{gamma} (KA = 0.004 ± 0.004). 2) There is a decrease in KA within the ALF sequence itself in the order of region II > region I > region IV, with a high value of 0.23 ± 0.022 (region II) and a low value of 0.021 ± 0.012 (region IV). The KA/KS ratio is much higher in region II of ALF (0.24) compared with region I (0.035) and region IV (0.040), or with TFIIA{alpha}/ß (0.033). The relatively high KA value in region II of ALF suggests relatively weak selection against nucleotide substitutions that cause amino acid changes.

In a comparison of 47 human and mouse genes [34] an average rate of 3.51 x 10-9 synonymous substitutions per site per year (rS) was calculated (assuming a divergence time of 80 million years), and the values for individual genes ranged from 1.57 x 10-9 to 6.39 x 10-9. An average rate of 0.74 x 10-9 nonsynonymous substitutions per site per year (rA) was also calculated and individual values ranged from 0.00 x 10-9 to 3.06 x 10-9. In comparison, the rates calculated for TFIIA{alpha}/ß were about average (rS = 3.3 x 10-9 and rA = 0.11 x 10-9), whereas the rates for ALF were slightly higher (rS = 6.2 x 10-9 and rA = 1.0 x 10-9).

These data show that the human and mouse ALF sequences, and region II in particular, are more divergent than TFIIA{alpha}/ß, TFIIA{gamma}, TBP, or TRF2. Although the physiological significance of this observation is currently unknown, the results indicate that the ALF gene has experienced a more rapid mutation rate, a reduced selection pressure, or both. Interestingly, other genes whose products are related to male reproductive function also diverge rapidly (see Discussion).

Genomic Structures of Human ALF, SBLF, TFIIA{alpha}/ß, and TFIIA{gamma}

Using ALF as a query of the htgs database at NCBI, a BAC clone that contained a complete ALF gene was identified. This gene is composed of nine exons (exon 1, 5'-UTR and aa 1–7; exon 2, aa 8–41; exon 3, aa 42–82; exon 4, aa 83–101; exon 5, aa 102–129; exon 6, aa 130–326; exon 7, aa 327–413; exon 8, aa 414–443; and exon 9, aa 444–478 and 3'-UTR; Fig. 2A).

Surprisingly, the clone noted above also contains a human Stoned B/µ-chain-like gene composed of four exons (exon 1, 5'-UTR; exon 2, aa 1–643; exon 3, aa 644–711; and exon 4, aa 712–736 and 3'-UTR). We denote this gene as ‘Stoned B-Like Factor’, or SBLF. The first three exons of the SBLF gene are identical to those present at the 5' end of SALF. However, several lines of evidence suggest that the normal transcript from this gene is not SALF, but is a 6.5-kb transcript reported earlier (RNA 6.5) [4]. Partial versions of this transcript have been identified in previous cloning experiments [4], by BLAST searches of human ESTs (e.g., GenBank accession number AI734225), and by PCR isolation of mouse SBLF clones from a mouse embryonic cDNA library (data not shown). Together with an examination of the SBLF-containing BAC clone, the results of these experiments show that SBLF encodes a 736-amino acid protein whose C-terminus extends 24 amino acids (VEIEKKWIKIDGEDPDKIGDCITQ) beyond the point at which SBLF and ALF are joined in SALF.

A gene for TFIIA{alpha}/ß was also identified in the htgs database. Like ALF, this gene also contains nine exons (exon 1, 5'-UTR and aa 1–10; exon 2, aa 11–44; exon 3, aa 45–112; exon 4, aa 113–134; exon 5, aa 135–159; exon 6, aa 160–204; exon 7, aa 205–311; exon 8, aa 312–341; and exon 9, aa 342–376 and 3'-UTR; Fig. 2B). Several exons divide the conserved regions of the ALF and TFIIA{alpha}/ß proteins at similar points; namely, exons 1–2, 2–3, 7–8, and 8–9 (Fig. 2, A and B). It is also important to note that the 5'- and 3'-UTRs have not been defined in the SBLF and TFIIA{alpha}/ß genes because cDNA clones for the full-length (6.0–7.0 kb) SBLF and TFIIA{alpha}/ß mRNAs have not been isolated. Thus the numbering of exons in these two genes is tentative.

A complete gene for TFIIA{gamma} was also identified, and is composed of five exons (exon 1, 5'-UTR; exon 2, 5'-UTR and aa 1–24; exon 3, aa 25–59; exon 4, aa 60–101; and exon 5, aa 102–109 and 3'-UTR; Fig. 2C). The intron-exon junctions identified in all four genes are flanked by splice donor (GT) or splice acceptor (AG) sequences, as expected (data not shown).

Up-Regulation of GTF Expression in Mouse Testis

In mammals, particular types of male germ cells initially appear at specific times during postnatal development [35]. In mice, for example, spermatogonia appear at postnatal Day 6, primary spermatocytes between postnatal Days 10 to 18, secondary spermatocytes at Postnatal Day 18, and haploid round spermatids at postnatal Day 20, followed by a 2-wk maturation process that yields mature spermatozoa. To determine whether ALF is expressed during germ cell differentiation, and to compare its pattern of expression with other GTFs, we examined testis RNA prepared from mice that were between 6 and 40 days old.

Northern blotting experiments using an ALF-specific probe showed no expression up to and including Day 12 (Fig. 3, lanes 1–3). This result suggests that ALF is not expressed in spermatogonia or in somatic cells of the testis. Rather, ALF expression was first detected on Postnatal Day 14 (lane 4), and mRNA levels continued to increase through Day 40 (compare lane 4 to lane 11). Expression at Day 14 coincides with the appearance of primary spermatocytes in the pachytene stage of meiosis [35], and the increase in RNA at later time points suggests continued expression in haploid spermatids.



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  		FIG. 3. A) Expression of GTFs during postnatal testis development in mouse. Northern blots containing RNA from the testes of mice at various days after birth (6, 10, 12, 14, 16, 18, 20, 22, 24, 27–30, and 40 days) were hybridized with the indicated probes (ALF, TFIIA{alpha}/ß, TFIIA{gamma}, TBP, TRF2, and TFIIIB90). B) Northern blot hybridization with probes for ß-actin and protamine-1. C) RT-PCR analysis using RNAs from male germ cells purified by Staput cell sedimentation. Primers for TFIIA{alpha}/ß, ALF, protamine-1, and ß-actin were used to detect the presence of the corresponding mRNAs in adult testis (lane 1), testis from 6-day-old animals (lane 2), purified pachytene spermatocytes (lane 3), round spermatids (lane 4), and elongating spermatids (lane 5)

We tested several other GTFs in this assay, including TFIIA{alpha}/ß, TFIIA{gamma}, TBP, and TRF2. These genes, unlike ALF, are expressed as early as Day 6 (Fig. 3A, lanes 1–3), probably because their gene products are required in all somatic cells, including those of the testis. However, the RNA level for each of these genes was distinctly elevated on Postnatal Day 14 (lane 4), and increased thereafter (lanes 5-11). In the case of TFIIA{alpha}/ß, two transcripts were detected (6.5 and 2.0 kb), possibly due to testis-specific transcription initiation or RNA processing. TBP shows expression during Days 6-12 (Fig. 3A, lanes 1–3) and is increased dramatically from Day 14 onward (lanes 4–11), in agreement with previous reports [3, 6]. Two TBP-related RNA species were observed (1.8 and 1.7 kb), possibly due to the use of alternative initiation sites [36]. In addition, we observed that an RNA polymerase III factor, TFIIIB90, was expressed during Days 6-12 (lanes 1–3) and was also increased from Day 14 onward (lanes 4–11). RT-PCR experiments revealed that the RNA polymerase I factor, TAFI 48, was also up-regulated between Days 12 and 14, whereas UBF was constant from Day 6 to Day 40 (data not shown). Overall, these results reveal a simultaneous and apparently coordinated increase in many GTFs during male germ cell differentiation, and include components from all three eukaryotic RNA polymerase systems.

To show that similar amounts of RNA were loaded onto these gels we normalized the blots using ß-actin (Fig. 3B). The results show equivalent levels of a 2.1-kb transcript in all lanes, with a smaller 2.0-kb band appearing from Day 24 onward (lanes 9-11). We also used a protamine-1 probe to demonstrate regulation of a gene expressed later in germ cell differentiation [37]. The results show a characteristic increase in expression of a 0.6-kb transcript from Days 22 to 24 through Day 40 (Fig. 3B, lanes 8–11).

Expression of ALF and TFIIA{alpha}/ß in Male Germ Cells

Northern blotting analysis of ALF expression during postnatal testis development suggested that ALF is a germ cell-specific counterpart of TFIIA{alpha}/ß. It remained possible, however, that ALF was actually expressed in somatic cells of the testis in a temporal pattern that paralleled the development of haploid germ cells. To identify specific cell types that express ALF and TFIIA{alpha}/ß in adult testis, we analyzed expression in germ cell fractions separated by Staput cell sedimentation [23–25]. The amount of first-strand cDNAs used in these experiments was normalized based on the results of RT-PCRs using ß-actin-specific primers (Fig. 3C, lower panel, lanes 1–5). PCRs with ALF-specific primers were then used to detect ALF expression in adult whole testis (Fig. 3C, lane 1) pachytene spermatocytes (lane 3), round spermatids (lane 4), and elongating spermatids (lane 5), but not in whole testis isolated at Postnatal Day 6 (lane 2). Likewise, PCRs with TFIIA{alpha}/ß-specific primers showed that TFIIA{alpha}/ß is also expressed in pachytene spermatocytes and spermatids (lanes 3–5), a result that suggests that ALF and TFIIA{alpha}/ß could be present in the same cells. Finally, reactions with protamine-1-specific primers were used to verify the characteristic expression of this gene in round and elongating spermatids (lanes 4 and 5).

In Situ Hybridization Analysis of ALF Expression in Mouse Testis

In situ hybridization was used to directly visualize the sites of ALF expression in testis. In these experiments, ALF mRNA was not detected in somatic cells of the testis or in spermatogonia. Instead, the antisense probe gave a strong signal in elongated spermatids of the seminiferous tubules at stages IV to VIII (Fig. 4). Certain tubules gave no signal (yellow arrowheads), whereas tubules containing round and elongating spermatids gave a more punctate signal (white arrows). This is presumably because tubules within a given section contain germ cells at distinct stages of development. No signal was detected in testis harvested at postnatal Day 14 (data not shown), suggesting that the level of expression at this time, although detectable by Northern analysis (Fig. 3), is too low to be seen by in situ hybridization. Neither the human nor the mouse sense probes detected ALF expression (Fig. 4 and data not shown). Collectively, these studies indicate that ALF and TFIIA{alpha}/ß mRNAs are present in meiotic and postmeiotic germ cells in the testis.



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  		FIG. 4. ALF is expressed in seminiferous tubules of mouse testis. Testes were harvested at 10 wk of age and used for in situ hybridization analyses as described in Materials and Methods. The top two panels show darkfield views of in situ hybridizations with sense (S) and antisense (AS) human ALF probes. The lower four panels show brightfield (left) and darkfield (right) views of in situ hybridizations with the mouse ALF antisense probe. Arrows indicate a tubule in which ALF expression was not detected, and arrowheads show a tubule containing round and elongating spermatids with a more punctate signal. The magnification is indicated to the left

Chromosomal Location of ALF, SBLF, and TFIIA{alpha}/ß

To determine the chromosomal location of ALF, SBLF, and TFIIA{alpha}/ß, genomic PCR was performed on a panel of human-hamster and human-mouse chromosomal hybrids. The ALF-specific primers amplified a 270-bp product in the SM852 cell line (Fig. 5A, top panel, lane 15), while the SBLF-specific primers amplified a 173-bp product (Fig. 5A middle panel, lane 15). As SM852 contains only human chromosome 2, both genes must be on this chromosome. These results are consistent with the assignment of ALF to the short arm of chromosome 2 between markers D2S119 and D2S337 (Locus ID 11036), and with our identification of the ALF and SBLF genes in a human chromosome 2 database.



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  		FIG. 5. A) ALF, SBLF, and TFIIA{alpha}/ß sequences were amplified from a panel of polychromosomal hybrid cell lines. Primers specific for ALF (top panel) amplified a product of 270 bp in line SM852 (lane 15), which contains human chromosome 2. Primers specific for SBLF (middle panel) amplified a product of 173 bp, also in SM852 (lane 15). Primers specific for TFIIA{alpha}/ß amplified a product in lanes 9, 11, 12, 16, 17, and 18. Discordancy analysis showed this signal correlated with the presence of human chromosome 14. Lanes 1–3 in all panels show control reactions using hamster, human, and mouse DNAs, respectively. B) Human genomic sequences that flank the SBLF and ALF regions in SALF reveal consensus splicing signals. Line 1 shows the sequence at the 5' end of the testis-specific ALF cDNA. Line 2 shows the same region in the SALF cDNA. The junction is indicated by the gap in the sequence. Line 3 shows the genomic sequence downstream of testis-specific ALF exon 1. Line 4 shows the genomic sequence upstream of ALF exon 2. An Alu-type element followed by an unusual (TAAA)10 repeat is found upstream of the splice junction. Line 5 shows the genomic sequence downstream of SBLF exon 3. Genomic sequences are capitalized. C) Model for the formation of SALF suggests that the testis-specific ALF gene is downstream of a ubiquitously expressed SBLF gene. The model suggests a pre-mRNA spanning these two otherwise independent transcription units is spliced between exon 3 of the upstream gene and exon 2 of the downstream gene to form the chimeric SALF transcript. The number of exons in the SBLF and ALF genes and sizes shown are based on analysis of human genomic DNA sequences. The exact size of exon 4 of SBLF has not been determined and is shown as an open, dashed box

Experiments performed with TFIIA{alpha}/ß-specific primers produced a 2.7-kb product derived from hamster DNA and a 2.5-kb human-specific fragment that appeared only in somatic cell hybrids that contained human chromosome 14 (Fig. 5A, lower panel, lanes 9, 11, 12, 16, 17, and 18). Again, this result is consistent with our identification of the TFIIA{alpha}/ß gene in a human chromosome 14 database.

Model for the Formation of SALF

SALF is a chimeric human transcript [4] that contains sequences that are identical to the SBLF and ALF genes. Because of its unusual chimeric structure, we wanted to evaluate how the SALF transcript might be formed. To address this question, we cloned genomic DNA adjacent to each of the three adjoining cDNA sequences shown in lines 1 and 2 of Figure 5B. As shown in lines 3–5, there is a splice donor (GT; lines 3 and 5) or a splice acceptor (AG) (line 4) site at the point at which the genomic and cDNA sequences meet. These results indicate that the junctions in the ALF and SALF cDNAs are defined by the boundaries of corresponding exons (lines 3–5). The sequences identified in these experiments match the genomic sequences identified in database searches. Interestingly, the intron sequence near the junction in line 4 resembles an Alu-type repetitive element, and contains a (TAAA)10 repeat [38].

Based on these results, together with the chromosomal localization and genomic sequence analysis of SBLF and ALF, we propose a model for SALF formation. According to the model, splicing of a putative pre-mRNA transcript that contains both genes forms the chimeric SALF cDNA (Fig. 5C and Discussion).

DISCUSSION

Germ Cell-Specific Expression of Transcription Factors

In this study we show that ALF is a male germ cell-specific counterpart of TFIIA{alpha}/ß. In particular, expression of ALF begins in testis at Postnatal Day 14, corresponding to the pachytene stage of meiosis. Furthermore, transcript levels increase through Day 40, suggesting that haploid spermatids continue to express this gene. These conclusions are supported by RT-PCR experiments using RNA from isolated germ cells, and by in situ hybridization studies that show ALF mRNA is present in elongated spermatids within the seminiferous tubules. Similarly, TBP was also up-regulated at Postnatal Day 14, consistent with earlier reports showing expression in early pachytene cells [3, 6] and with the immunological detection of the TBP protein in haploid spermatids [3].

We also show that the RNA polymerase II factors TFIIA{alpha}/ß, TFIIA{gamma} and TRF2, as well as the RNA polymerase III factor TFIIIB90, are up-regulated during germ cell differentiation in mouse testis. Because these genes are expressed in a pattern that is very similar to that observed with ALF and TBP, we predict they are also enriched in meiotic and haploid germ cells. The current data therefore suggest a widespread and apparently coordinated up-regulation of GTF genes in male germ cells. It seems likely that this up-regulation will be directed by one or more pathways or processes that are activated only in male germ cells.

The transcription factors tested here and elsewhere fit into at least two categories. The first category includes those expressed in all somatic cells, including those of the testis, and which are also present in male germ cells. Examples include TBP, TRF2, TFIIA{alpha}/ß, TFIIA{gamma}, TFIIB [3], and TFIIIB90. The second category consists of genes whose expression is primarily restricted to male germ cells. Examples include ALF, the elongation factor SII-T1 [17] and Elongin A2 [18] genes, and perhaps the gonad- and CNS-specific Drosophila factor, TRF1 [8]. Factors whose expression is decreased or off in male germ cells have not been identified here, but would fall into a hypothetical third category.

A key question to be answered is the relationship between GTF expression and the biological functions of testis (reviewed in [39, 40]). It is possible that high levels of GTFs are required in order to express sufficient levels of genes needed for meiosis and spermatogenesis. Alternatively, the observation that some testis RNAs, for instance TBP [36], initiate at promoters that are not used in somatic cells raises the idea that transcription is somewhat indiscriminate in this tissue. This characteristic may be due to increased GTF expression, or to increased accessibility to genomic DNA as germ cell chromatin is remodeled for packaging into spermatozoa.

A hypothetical role for testis-specific GTF-related factors such as ALF is to regulate a particular subset of genes. In the case of the Drosophila TBP-related factor TRF1, this idea is supported by localization of the protein to distinct sites on polytene chromosomes [9]. In addition, mutations in the trf gene cause neurological defects and male sterility, demonstrating that TRF1 is important for CNS and gonadal function [8]. Likewise, TBP-associated factors (TAFs) in TFIID, as well as TFIIA, have been implicated in the regulation of genes that control cell cycle progression [41–44]. Similarly, ALF may have a specialized role in the expression of genes that control progression through meiosis, or those that control germ cell development.

In some cases the level of GTF mRNA in testis is much higher than the level of the corresponding protein. For instance, the TBP protein is between twofold to 11-fold higher in testis compared with other tissues, whereas its mRNA is estimated to be between 40- and 1000-fold higher [3, 6, 7]. Likewise, transcripts for the TBP-related factor, TRF2, are expressed at high levels in mouse testis [11], but immunoblotting data show that the level of TRF2 protein is comparable to or lower than in other tissues [45]. A partial explanation for this discrepancy is suggested from the observation that testis-specific transcripts from the TBP gene are underrepresented among polyribosomal RNAs [7]. Thus, perhaps some RNA produced from the GTF genes is packaged into ribonucleoprotein complexes that repress or delay translation [46]. Potentially related, the protein levels for the {alpha} subunit of TFIIE, the large subunit of RNA polymerase II, and the 30-, 55-, 100-, and 135-kDa TAFII subunits show only a modest increase in testis [3, 7, 45].

Evolution of ALF and TFIIA{alpha}/ß

A comparison between human and mouse counterparts of TBP, TRF2, TFIIA{gamma}, and TFIIA{alpha}/ß shows that these sequences are 95%–100% similar. In contrast, the testis-specific human and mouse ALF sequences are much more divergent (77% similarity), mainly due to differences in region II (68% similarity). Rapid evolution among genes involved in male reproductive physiology such as the sex-determining SRY gene [47, 48], the Pem homeobox transcription factor gene [49], and others [50–52], has previously been noted. In addition, gene families that contain members expressed in distinct somatic and germ-cell patterns (like ALF and TFIIA{alpha}/ß) often show a similar pattern. For example, the human somatic transcription elongation factor, TFIIS, is 96% identical to its mouse counterpart, whereas the testis specific form (SII-T1) is 89% identical [53]. Likewise, the human somatic phosphoglycerate kinase (PGK1) gene is 98% similar to its mouse counterpart, whereas the testis-specific isoform (PGK2) is 86% similar [54]. Finally, the human lactate dehydrogenase (LDH) A isoform is 95% similar to its mouse counterpart, while the testis-specific LDH C isoform is only 73% similar [55]. The biochemical mechanisms that underlie the greater rate of evolution for these and other male reproductive genes are not understood, and it is unclear whether such divergence confers some reproductive advantage.

Structure of SALF

In this report we also sought to evaluate possible models that might explain the formation of the chimeric SALF transcript. One such possibility is that SALF is a product of two separate but adjacent human genes. Evidence for this idea is based on the fact that the 5' and 3' ends of SALF are unrelated, and on the observation that a probe from the 5' end of SALF detects a 6.5-kb mRNA in all tissues while a probe from the 3' end of SALF detects a 1.8-kb mRNA in testis (ALF) [4]. We have shown here that genomic sequences that border the junction within the ALF and SALF cDNAs contain putative splice donor (GT) and splice acceptor (AG) signals (Fig. 5A). Furthermore, PCR analysis of polychromosomal hybrid DNAs shows that the ALF and SBLF genes are both on chromosome 2. These results are substantiated by the intriguing discovery that complete ALF and SBLF genes are present on a single BAC clone.

Based on these data we propose that SALF is formed from a pre-mRNA transcript that initiates at an upstream SBLF gene and continues into the downstream ALF gene (Fig. 5C). The third exon of the SBLF gene is then spliced to the second exon of ALF to produce SALF. The model predicts that expression of SALF depends on the same regulatory elements that control expression of SBLF. This idea is consistent with the observation that a rare transcript detected with an ALF probe in nontestis tissues varies in proportion to the levels of the 6.5-kb SBLF transcript [4]. If correct, the model provides an unusual example of a eukaryotic pre-mRNA transcript that spans two independent genes. However, the model does not address whether the 1182-amino acid SALF polypeptide is physiologically important.

Conclusion

The results reported here demonstrate that ALF is a male germ cell-specific counterpart of TFIIA{alpha}/ß, and that an increase in the expression of many GTFs occurs during male germ cell development. It will be of interest to elucidate the factors and mechanisms that direct this expression, and to determine the relationship or relationships between GTF function and the cellular and chromosomal changes that occur during spermatogenesis.

ACKNOWLEDGMENTS

The sequences reported in this paper have been submitted to GenBank under the following accession numbers: rat TFIIA{gamma} (AF000944), rat TFIIA{alpha}/ß (AF000943), mouse ALF (AF250835), and mouse TFIIA{alpha}/ß (AF250834). The authors thank the Washington University and Genoscope sequencing centers for making human genomic sequence available prior to publication.

FOOTNOTES

First decision: 31 May 2000.

1 This work was supported by grants from the American Cancer Society and the Welch Foundation (J.D.), and by NIH grant DK54480 (K.L.P.) Back

2 Correspondence: Jeff DeJong, The University of Texas at Dallas, Dept. of Molecular and Cell Biology, 2601 N. Floyd Rd., Richardson, TX 75080. FAX: 972 883 2409; dejong{at}utdallas.edu Back

Accepted: September 6, 2000.

Received: May 11, 2000.

REFERENCES

  1. Roeder RG. The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem Sci 1996; 21:327–335.[CrossRef][Medline]
  2. Orphanides G, Lagrange T, Reinberg D. The general transcription factors of RNA polymerase II. Genes Dev 1996; 10:2657–2683.[Free Full Text]
  3. Schmidt EE, Schibler U. High accumulation of components of the RNA polymerase II transcription machinery in rodent spermatids. Development 1995; 121:2373–2383.[Abstract]
  4. Upadhyaya AB, Lee SH, DeJong J. Identification of a general transcription factor TFIIA{alpha}/ß homolog selectively expressed in testis. J Biol Chem 1999; 274:18040–18048.[Abstract/Free Full Text]
  5. Ozer J, Moore PA, Lieberman PM. A testis-specific transcription factor IIA (TFIIA{tau}) stimulates TATA-binding protein-DNA binding and transcription activation. J Biol Chem 2000; 275:122–128.[Abstract/Free Full Text]
  6. Persengiev SP, Robert S, Kilpatrick DL. Transcription of the TATA binding protein gene is highly up-regulated during spermatogenesis. Mol Endocrinol 1996; 10:742–747.[Abstract]
  7. Schmidt EE, Schibler U. Developmental testis-specific regulation of mRNA levels and mRNA translation efficiencies for TATA-binding protein mRNA isoforms. Dev Biol 1997; 184:138–149.[CrossRef][Medline]
  8. Crowley TE, Hoey T, Liu J-K, Jan YN, Jan LY, Tjian R. A new factor related to TATA-binding protein has highly restricted expression patterns in Drosophila. Nature 1993; 361:557–561.[CrossRef][Medline]
  9. Hansen S, Takada S, Jacobson RH, Lis JT, Tjian R. Transcription properties of a cell type-specific TATA-binding protein, TRF. Cell 1997; 91:71–83.[CrossRef][Medline]
  10. Wieczorek E, Brand M, Jacq X, Tora L. Function of TAFII-containing complex without TBP in transcription by RNA polymerase II. Nature 1998; 393:187–191.[CrossRef][Medline]
  11. Ohbayashi T, Makino Y, Tamura T-A. Identification of a mouse TBP-like protein (TLP) distantly related to the Drosophila TBP-related factor. Nucleic Acids Res 1999; 27:750–755.[Abstract/Free Full Text]
  12. Shimada M, Ohbayashi T, Ishida M, Nakadai T, Makino Y, Aoki T, Kawata T, Suzuki T, Matsuda Y, Tamura T-A. Analysis of the chicken TBP-like protein (tlp) gene: evidence for a striking conservation of vertebrate TLPs and for a close relationship between vertebrate tbp and tlp genes. Nucleic Acids Res 1999; 27:3146–3152.[Abstract/Free Full Text]
  13. Rabenstein MD, Zhou S, Lis JT, Tjian R. TATA box-binding protein (TBP)-related factor 2 (TRF2), a third member of the TBP family. Proc Natl Acad Sci U S A 1999; 96:4791–4796.[Abstract/Free Full Text]
  14. Maldonado E. Transcriptional functions of a new mammalian TATA-binding protein-related factor. J Biol Chem 1999; 274:12963–12966.[Abstract/Free Full Text]
  15. Moore PA, Ozer J, Salunek M, Jan G, Zerby D, Campbell S, Lieberman PM. A human TATA binding protein-related protein with altered DNA binding specificity inhibits transcription from multiple promoters and activators. Mol Cell Biol 1999; 19:7610–7620.[Abstract/Free Full Text]
  16. Teichmann M, Wang Z, Martinez E, Tjernberg A, Zhang D, Vollmer F, Chait BT, Roeder RG. Human TATA-binding protein-related factor 2 (hTRF2) stably associates with hTFIIA in HeLa cells. Proc Natl Acad Sci U S A 1999; 96:13720–23725.[Abstract/Free Full Text]
  17. Xu Q, Nakanishi T, Sekimizu K, Natori S. Cloning and identification of a testis-specific transcription elongation factor S-II. J Biol Chem 1994; 269:3100–3103.[Abstract/Free Full Text]
  18. Aso T, Yamazaki K, Amimoto K, Kuroiwa A, Higashi H, Matsuda Y, Kitajima S, Hatakeyama M. Identification and characterization of elongin A2, a new member of the elongin family of transcription elongation factors, specifically expressed in testis. J Biol Chem 2000; 275:6546–6552.[Abstract/Free Full Text]
  19. Andrews J, Smith M, Merakovsky J, Coulson M, Hannan F, Kelly LE. The stoned locus of Drosophila melanogaster produces a dicistronic transcript and encodes two distinct polypeptides. Genetics 1996; 143:1699–1711.[Abstract]
  20. Hirst J, Robinson MS. Clathrins and adaptors. Biochim Biophys Acta 1998; 1404:173–193.[Medline]
  21. DeJong J, Bernstein R, Roeder RG. Human general transcription factor TFIIA: characterization of a cDNA encoding the small subunit and requirement for basal and activated transcription. Proc Natl Acad Sci U S A 1995; 92:3313–3317.[Abstract/Free Full Text]
  22. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156–159.[Medline]
  23. Lam DMK, Furrer R, Bruce WR. The separation, physical characterization, and differentiation kinetics of spermatogonial cells of the mouse. Proc Natl Acad Sci U S A 1970; 65:192–199.[Abstract/Free Full Text]
  24. Meistrich ML, Bruce WR, Clermont Y. Cellular composition of fractions of mouse testis cells following velocity sedimentation separation. Exp Cell Res 1973; 79:213–227.[CrossRef][Medline]
  25. Meistrich ML. Separation of spermatogenic cells and nuclei from rodent testes. Methods Cell Biol 1977; 15:15–54.[Medline]
  26. Kang JJ, Auble DT, Ranish JA, Hahn S. Analysis of the yeast transcription factor TFIIA: distinct functional regions and a polymerase II-specific role in basal and activated transcription. Mol Cell Biol 1995; 15:1234–1243.[Abstract]
  27. Tan S, Hunziker Y, Sargent DF, Richmond TJ. Crystal structure of a yeast TFIIA/TBP/DNA complex. Nature 1996; 381:127–134.[CrossRef][Medline]
  28. Geiger JH, Hahn S, Lee S, Sigler PB. Crystal structure of the yeast TFIIA/TBP/DNA complex. Science 1996; 272:830–836.[Abstract]
  29. DeJong J, Roeder RG. A single cDNA, hTFIIA/{alpha}, encodes both the p35 and p19 subunits of human TFIIA. Genes Dev 1993; 7:2220–2234.[Abstract/Free Full Text]
  30. Ma D, Watanabe H, Mermelstein F, Admon A, Oguri K, Sun X, Wada T, Imai T, Shiroya T, Reinberg D, Handa H. Isolation of a cDNA encoding the largest subunit of TFIIA reveals functions important for activated transcription. Genes Dev 1993; 7:2246–2257.[Abstract/Free Full Text]
  31. Ozer J, Moore PA, Bolden AH, Lee A, Rosen CA, Leiberman P. Molecular cloning of the small ({gamma}) subunit of human TFIIA reveals functions critical for activated transcription. Genes Dev 1994; 8:2324–2335.[Abstract/Free Full Text]
  32. Sun X, Ma D, Sheldon M, Yeung K, Reinberg D. Reconstitution of human TFIIA activity from recombinant polypeptides: a role in TFIID-mediated transcription. Genes Dev 1994; 8:2336–2348.[Abstract/Free Full Text]
  33. Kumar S, Tamura K, Nei M. MEGA v. 1.01. University Park, PA: The Pennsylvania State University; 1993.
  34. Li W-H. Molecular Evolution. Sunderland, MA: Sinauer Associates, Inc, Publishers; 1997: 177–182.
  35. McCarrey JR. Development of the germ cell. In: Desjardins C, Ewing LL (eds.), Cell and Molecular Biology of the Testis. New York: Oxford University Press; 1993: 58–89.
  36. Schmidt EE, Ohbayashi T, Makino Y, Tamura T, Schibler U. Spermatid-specific overexpression of the TATA-binding protein gene involves recruitment of two potent testis-specific promoters. J Biol Chem 1997; 272:5326–5334.[Abstract/Free Full Text]
  37. Hecht NB, Bower PA, Waters SH, Yelick PC, Distel RJ. Evidence for haploid expression of mouse testicular genes. Exp Cell Res 1986; 164:183–190.[CrossRef][Medline]
  38. Economou EP, Bergen AW, Warren AC, Antonarakis SE. The polydeoxyadenylate tract of Alu repetitive elements is polymorphic in the human genome. Proc Natl Acad Sci U S A 1990; 87:2951–2954.[Abstract/Free Full Text]
  39. Hecht NB. Gene expression during the male germ cell development. In: Desjardins C, Ewing LL (eds.), Cell and Molecular Biology of the Testis. New York: Oxford University Press; 1993: 400–432.
  40. Eddy EM. Gene expression during mammalian meiosis. Curr Top Dev Biol 1995; 37:141–200.
  41. Ruppert S, Wang EH, Tjian R. Cloning and expression human TAFII250: a TBP-associated factor implicated in cell-cycle regulation. Nature 1993; 362:175–178.[CrossRef][Medline]
  42. Hisatake K, Hasegawa S, Takada T, Nakatani Y, Horikoshi M, Roeder RG. The p250 subunit of native TATA box-binding factor TFIID is the cell-cycle regulatory protein CCG1. Nature 1993; 362:179–181.[CrossRef][Medline]
  43. Apone LM, Virbasius C-MA, Reese JC, Green MR. Yeast TAFII90 is required for cell-cycle progression through G2/M but not for general transcription activation. Genes Dev 1996; 10:2368–2380.[Abstract/Free Full Text]
  44. Ozer J, Lezina LE, Ewing J, Audi S, Lieberman PM. Association of transcription factor IIA with TATA binding protein is required for transcriptional activation of a subset of promoters and cell cycle progression in Saccharomyces cerevisiae. Mol Cell Biol 1998; 18:2559–2570.[Abstract/Free Full Text]
  45. Perletti L, Dantonel J-C, Davidson I. The TATA-binding protein and its associated factors are differentially expressed in adult mouse tissues. J Biol Chem 1999; 274:15301–15304.[Abstract/Free Full Text]
  46. Kleene KC. Patterns of translational regulation in the mammalian testis. Mol Reprod Dev 1996; 43:268–281.[CrossRef][Medline]
  47. Whitfield LS, Lovell-Badge R, Goodfellow PN. Rapid sequence evolution of the mammalian sex-determining gene SRY. Nature 1993; 364:713–715.[CrossRef][Medline]
  48. Tucker PK, Lundrigan BL. Rapid evolution of the sex determining locus in Old World mice and rats. Nature 1993; 364:715–717.[CrossRef][Medline]
  49. Maiti S, Doskow J, Sutton K, Nhim RP, Lawlor DA, Levan K, Lindsey JS, Wilkinson MF. The Pem homeobox gene: rapid evolution of the homeodomain, X chromosomal localization, and expression in reproductive tissue. Genomics 1996; 34:304–316.[CrossRef][Medline]
  50. Ferris PJ, Pavlovic C, Fabry S, Goodenough UW. Rapid evolution of sex-related genes in Chlamydomonas. Proc Natl Acad Sci U S A 1997; 94:8634–8639.[Abstract/Free Full Text]
  51. Civetta A, Singh RS. Sex-related genes, directional sexual selection, and speciation. Mol Biol Evol 1998; 15:901–909.[Abstract]
  52. Wyckoff GJ, Wang W, Wu C-I. Rapid evolution of male reproductive genes in the descent of man. Nature 2000; 403:304–309.[CrossRef][Medline]
  53. Umehara T, Kida S, Yamamoto T, Horikoshi M. Isolation and characterization of a cDNA encoding a new type of human transcription elongation factor S-II. Gene 1995; 167:297–302.[CrossRef][Medline]
  54. McCarrey JR. Molecular evolution of the human Pgk-2 retroposon. Nucleic Acids Res 1990; 18:949–955.[Abstract/Free Full Text]
  55. Millan JL, Driscoll CE, LeVan KM, Goldberg E. Epitopes of human testis-specific lactate dehydrogenase deduced from a cDNA sequence. Proc Natl Acad Sci U S A 1987; 84:5311–5315.[Abstract/Free Full Text]



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H. Wang, J. T. San Agustin, G. B. Witman, and D. L. Kilpatrick
Novel Role for a Sterol Response Element Binding Protein in Directing Spermatogenic Cell-Specific Gene Expression
Mol. Cell. Biol., December 15, 2004; 24(24): 10681 - 10688.
[Abstract] [Full Text] [PDF]

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 Biol. Reprod.Home page
S. Han, W. Xie, S. H. Kim, L. Yue, and J. DeJong
A Short Core Promoter Drives Expression of the ALF Transcription Factor in Reproductive Tissues of Male and Female Mice
Biol Reprod, September 1, 2004; 71(3): 933 - 941.
[Abstract] [Full Text] [PDF]

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 Mol. Endocrinol.Home page
D. De Cesare, G. M. Fimia, S. Brancorsini, M. Parvinen, and P. Sassone-Corsi
Transcriptional Control in Male Germ Cells: General Factor TFIIA Participates in CREM-Dependent Gene Activation
Mol. Endocrinol., December 1, 2003; 17(12): 2554 - 2565.
[Abstract] [Full Text] [PDF]

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 J. Biol. Chem.Home page
S. Han, W. Xie, S. R. Hammes, and J. DeJong
Expression of the Germ Cell-specific Transcription Factor ALF in Xenopus Oocytes Compensates for Translational Inactivation of the Somatic Factor TFIIA
J. Biol. Chem., November 14, 2003; 278(46): 45586 - 45593.
[Abstract] [Full Text] [PDF]

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S.-i. Kashiwabara, J. Noguchi, T. Zhuang, K. Ohmura, A. Honda, S. Sugiura, K. Miyamoto, S. Takahashi, K. Inoue, A. Ogura, et al.
Regulation of Spermatogenesis by Testis-Specific, Cytoplasmic Poly(A) Polymerase TPAP
Science, December 6, 2002; 298(5600): 1999 - 2002.