Upstream Control Element Research Articles

Ribosomal DNA methylation in human and mouse oocytes increases with age.

An age-dependent increase in ribosomal DNA (rDNA) methylation has been observed across a broad spectrum of somatic tissues and the male mammalian germline. Bisulfite pyrosequencing (BPS) was used to determine the methylation levels of the rDNA core promoter and the rDNA upstream control element (UCE) along with two oppositely genomically imprinted control genes (PEG3 and GTL2) in individual human germinal vesicle (GV) oocytes from 90 consenting women undergoing fertility treatment because of male infertility. Apart from a few (4%) oocytes with single imprinting defects (in either PEG3 or GTL2), the analyzed GV oocytes displayed correct imprinting patterns. In 95 GV oocytes from 42 younger women (26-32 years), the mean methylation levels of the rDNA core promoter and UCE were 7.4±4.0% and 9.3±6.1%, respectively. In 79 GV oocytes from 48 older women (33-39 years), methylation levels increased to 9.3±5.3% (P = 0.014) and 11.6±7.4% (P = 0.039), respectively. An age-related increase in oocyte rDNA methylation was also observed in 123 mouse GV oocytes from 29 4-16-months-old animals. Similar to the continuously mitotically dividing male germline, ovarian aging is associated with a gain of rDNA methylation in meiotically arrested oocytes. Oocytes from the same woman can exhibit varying rDNA methylation levels and, by extrapolation, different epigenetic ages.

P–528 rDNA methylation of human oocytes of women undergoing intracytoplasmic sperm injection (ICSI) increases with maternal age

Abstract Study question Is there a correlation between the age of women undergoing ICSI and methylation pattern of rDNA core promoter and upstream control element in immature human oocytes? Summary answer Methylation levels of the upstream control element and the rDNA core promoter in immature human oocytes increase with age of women undergoing ICSI. What is known already Methylation of ribosomal DNA (rDNA) in germ cells regulates temporary and spatially highly coordinated nucleolar activity, cellular metabolism, and thus developmental potential of the early embryo. Alterations of methylation pattern may therefore cause dysregulation of genes and signal cascades resulting in limited fertility. It has been shown that the methylation of sperm rDNA increases with the donor’s age. The positive correlation between sperm rDNA methylation and age has been conserved among mammals during evolution including humans and mice. In contrast to sperm, little is known about the methylome of human oocytes and its role in human reproduction. Study design, size, duration Consecutive women undergoing ICSI because of male subfertility were included. Patients with endometriosis, polycystic ovary syndrome, ovarian, uterine or breast cancer, as well as patients with an anti-Mullerian hormone level <1ng/ml were excluded. Immature oocytes (germinal vesicle; GV) collected during oocyte pick-up at the Fertility Centre Dortmund between 2018 and 2020 were examined. Participants/materials, setting, methods Cumulus-free GV oocytes which were not usable for ICSI were rinsed in phosphate buffer and stored at –20 °C until further investigation. Multiplex-PCR followed by singleplex-PCRs were carried out on the rDNA core promoter and upstream control element. Methylation levels were quantified by bisulphite pyrosequencing. Two oppositely imprinted genes (hPEG3 and hGTL2) were used as controls to ensure correct amplification and bisulphite conversion. Spearman’s-rank-order-correlation and Mann-Whitney-U-Test were used for statistical analysis. Main results and the role of chance For each GV oocyte, nine different Cytosine-phosphate-Guanine dinucleotides (CpGs) were quantified by bisulphite pyrosequencing for the rDNA core promoter and 26 different CpGs for the upstream control element (UCE). 120 human single oocytes from 60 women were analyzed. Connected statistical analysis was used if one patient had more than one oocyte. The age of the included women ranged from 26 to 40 years (mean±SD 33.5±3.2). Only oocytes which showed a correct methylation pattern for at least one imprinting control gene (hPEG3 and hGTL2) were considered for analysis. Mean methylation level ranged from 2–31% (mean±SD 8.7±5.5) of the analyzed CpGs for the rDNA core promoter and from 3–36% (mean±SD 11.4±7.1) CpGs for UCE. Spearman’s correlation analysis revealed that the methylation levels of the human oocyte rDNA core promoter and rDNA UCE significantly increased with the age of the donor (p < 0.05). Correlation coefficient for rDNA core promoter was r = 0.22 and for upstream control element r = 0.21. It is also interesting to note that different oocytes from the same donors can display enormous methylation variation. Regarding clinical parameters, no correlation was observed between the methylation pattern of the rDNA core promoter or UCE and the body mass index or smoking status, respectively. Limitations, reasons for caution Limitations of this study include difficulties in extrapolating the findings to the general population, because no data of women not undergoing ICSI are available. Only GV-oocytes were analyzed. Additional research is needed to clarify the effect of different methylation pattern with increasing female age and its role in human reproduction. Wider implications of the findings: We propose that the increase of rDNA methylation in male and female germ cells with advanced age directly or indirectly influences the regulation of nucleolar activity, cellular metabolism, and thus the developmental potential of the early embryo. This age-dependent epigenetic effect may result in decreased human fertility. Trial registration number NCT03565107

Development of a Virus-Based Reporter System for Functional Analysis of Plant rRNA Gene Promoter.

Reporter gene-based expression systems have been intensively used in plants for monitoring the activity of gene promoters. However, rRNA transcripts are unable to efficiently express a reporter gene due to a lack of a 5' cap. Because of this obstacle, plant rRNA gene promoters are less well characterized to this day. We developed a virus-based reporter system to characterize the Nicotiana benthamiana rRNA (NbrRNA) gene promoter. The system utilizes the cap-independent translation strategy of viral genomic mRNA and uses the virus-expressed green fluorescent protein (GFP) as an indicator of the rRNA gene promoter activity in virus-infected plants. Based on the reporter system, some characteristics of the N. benthamiana rRNA gene promoter were revealed. The results showed that the strength of the NbrRNA gene promoter was lower than that of the cauliflower mosaic virus (CaMV) 35S promoter, a well-characterized polymerase II promoter. The sequences between −77 and +42 are sufficient for the NbrRNA gene promoter-mediated transcription and the NbrRNA gene promoter may lack the functional upstream control element (UCE). Interestingly, NbrRNA gene promoter activity was increased when the 35S enhancer was introduced. An intron-excision mediated assay revealed that the NbrRNA gene promoter can be inefficiently used by RNA polymerase II in N. benthamiana cells. This virus-based reporter system is easier to operate and more convenient when compared with the previously Pol I promoter assays. And it offers a promising solution to analyzing the functional architecture of plant rRNA gene promoter.

Increasing methylation of sperm rDNA and other repetitive elements in the aging male mammalian germline.

In somatic cells/tissues, methylation of ribosomal DNA (rDNA) increases with age and age‐related pathologies, which has a direct impact on the regulation of nucleolar activity and cellular metabolism. Here, we used bisulfite pyrosequencing and show that methylation of the rDNA transcription unit including upstream control element (UCE), core promoter, 18S rDNA, and 28S rDNA in human sperm also significantly increases with donor's age. This positive correlation between sperm rDNA methylation and biological age is evolutionarily conserved among mammals with widely different life spans such as humans, marmoset, bovine, and mouse. Similar to the tandemly repeated rDNA, methylation of human α‐satellite and interspersed LINE1 repeats, marmoset α‐satellite, bovine alpha‐ and testis satellite I, mouse minor and major satellite, and LINE1‐T repeats increases in the aging male germline, probably related to their sperm histone packaging. Deep bisulfite sequencing of single rDNA molecules in human sperm revealed that methylation does not only depend on donor's age, but also depend on the region and sequence context (A vs. G alleles). Both average rDNA methylation of all analyzed DNA molecules and the number of fully (>50%) methylated alleles, which are thought to be epigenetically silenced, increase with donor's age. All analyzed CpGs in the sperm rDNA transcription unit show comparable age‐related methylation changes. Unlike other epigenetic aging markers, the rDNA clock appears to operate in similar ways in germline and soma in different mammalian species. We propose that sperm rDNA methylation, directly or indirectly, influences nucleolar formation and developmental potential in the early embryo.

Heterozygous De Novo UBTF Gain-of-Function Variant Is Associated with Neurodegeneration in Childhood

Ribosomal RNA (rRNA) is transcribed from rDNA by RNA polymerase I (Pol I) to produce the 45S precursor of the 28S, 5.8S, and 18S rRNA components of the ribosome. Two transcription factors have been defined for Pol I in mammals, the selectivity factor SL1, and the upstream binding transcription factor (UBF), which interacts with the upstream control element to facilitate the assembly of the transcription initiation complex including SL1 and Pol I. In seven unrelated affected individuals, all suffering from developmental regression starting at 2.5-7 years, we identified a heterozygous variant, c.628G>A in UBTF, encoding p.Glu210Lys in UBF, which occurred de novo inall cases. While the levels of UBF, Ser388 phosphorylated UBF, and other Pol I-related components (POLR1E, TAF1A, and TAF1C) remained unchanged in cells of an affected individual, the variant conferred gain of function to UBF, manifesting by markedly increased UBF binding to the rDNA promoter and to the 5'- external transcribed spacer. This was associated with significantly increased 18S expression, and enlarged nucleoli which were reduced in number per cell. The data link neurodegeneration in childhood with altered rDNA chromatin status and rRNA metabolism.

Angiogenin Stimulates Ribosomal RNA Transcription by Epigenetic Activation of the Ribosomal DNA Promoter

Angiogenin (ANG) undergoes nuclear translocation and promotes ribosomal RNA (rRNA) transcription thereby enhancing cell growth and proliferation. However, the mode of action of ANG in stimulating rRNA transcription is unclear. Here, we show that ANG enhances the formation of RNA polymerase I (Pol I) pre-initiation complex at the ribosomal DNA (rDNA) promoter. ANG binds at the upstream control element (UCE) of the promoter and enhances promoter occupancy of RNA Pol I as well as the selectivity factor SL1 components TAFI 48 and TAFI 110. We also show that ANG increases the number of actively transcribing rDNA by epigenetic activation through promoter methylation and histone modification. ANG binds to histone H3, inhibits H3K9 methylation, and activates H3K4 methylation as well as H4 acetylation at the rDNA promoter. These data suggest that one of the mechanisms by which ANG stimulates rRNA transcription is through an epigenetic activation of rDNA promoter.

Expression of Expanded CAG Transcripts Triggers Nucleolar Stress in Huntington's Disease

Polyglutamine (polyQ) diseases, including several types of spinocerebellar ataxias and Huntington's disease (HD), are dominantly inherited neurodegenerative disorders caused by the expansion of the glutamine-coding CAG repeat in the open reading frame of the disease gene. Apart from being translated to produce toxic elongated polyQ domain-containing disease proteins, transcribed expanded CAG RNAs per se also exert toxicity in polyQ degeneration. In the R6/2 HD transgenic mouse model, expanded mutant Huntingtin (Htt) transcripts were found to physically interact with nucleolin (NCL), a nucleolar protein that plays a crucial role in ribosome biogenesis. We further demonstrated that mutant Htt transcripts deprived NCL from binding onto the Upstream Control Element (UCE) of the ribosomal RNA (rRNA) promoter. This resulted in UCE hypermethylation which abolished the binding of the transcription factor Upstream Binding Factor to UCE and subsequently led to down-regulation of pre-45s rRNA transcription. We also found that the p53/mitochondria-dependent nucleolar stress cell death pathway was activated in polyQ diseases. Ribosomal RNA transcription dysfunction has been reported in other types of neurodegenerative disorders including Alzheimer's disease; it is anticipated that nucleolar stress is one common pathogenic signaling mechanism shared by different forms of neurodegeneration.

Trichomonas vaginalis ribosomal RNA: Identification and characterisation of the transcription promoter and terminator sequences

Trichomonas vaginalis is a parasitic protozoan of both medical and biological relevance. Transcriptional studies in this organism have focused mainly on type II pol promoters, whereas the elements necessary for transcription by polI or polIII have not been investigated. Here, with the aid of a transient transcription system, we characterised the rDNA intergenic region, defining both the promoter and the terminator sequences required for transcription. We defined the promoter as a compact region of approximately 180bp. We also identified a potential upstream control element (UCE) that was located 80bp upstream of the transcription start point (TSP). A transcription termination element was identified within a 34bp region that was located immediately downstream of the 28S coding sequence. The function of this element depends upon polarity and the presence of both a stretch of uridine residues (U's) and a hairpin structure in the transcript. Our observations provide a strong basis for the study of DNA recognition by the polI transcriptional machinery in this early divergent organism.

PARP-1 Expression in the Mouse is Controlled by an Autoregulatory Loop: PARP-1 Binding to an Upstream S/MAR Element and to a Novel Recognition Motif in its Promoter Suppresses Transcription

This work identifies central components of a feedback mechanism for the expression of mouse poly(ADP-ribose) polymerase-1 (PARP-1). Using the stress-induced duplex destabilization algorithm, multiple base-unpairing regions (BURs) could be localized in the 5′ region of the mouse PARP-1 gene ( muPARP-1). Some of these could be identified as scaffold/matrix-attachment regions (S/MARs), suggesting an S/MAR-mediated transcriptional regulation. PARP-1 binding to the most proximal element, S/MAR 1, and to three consensus motifs, AGGCC, in its own promoter (basepairs –956 to +100), could be traced by electrophoretic mobility-shift assay. The AGGCC-complementary GGCCT motif was detected by cis-diammine-dichloro platinum cross-linking and functionally characterized by the effects of site-directed mutagenesis on its performance in wild type (PARP-1 +/+) and PARP-1 knockout cells (PARP-1 -/-). Mutation of the central AGGCC tract at basepairs –554 to –550 prevented PARP-1/promoter interactions, whereby muPARP-1 expression became up-regulated. Transfection of a series of reporter gene constructs with or without S/MAR 1 (basepairs –1523 to –1007) and the more distant S/MAR 2 (basepairs –8373 to –6880), into PARP-1 +/+ as well as PARP-1 -/- cells, revealed an additional, major level of muPARP-1 promoter down-regulation, triggered by PARP-1 binding to S/MAR 1. We conclude that S/MAR 1 represents an upstream control element that acts in conjunction with the muPARP-1 promoter. These interactions are part of a negative autoregulatory loop.

TAF12 Recruits Gadd45a and the Nucleotide Excision Repair Complex to the Promoter of rRNA Genes Leading to Active DNA Demethylation

Many studies have detailed the repressive effects of DNA methylation on gene expression. However, the mechanisms that promote active demethylation are just beginning to emerge. Here, we show that methylation of the rDNA promoter is a dynamic and reversible process. Demethylation of rDNA is initiated by recruitment of Gadd45a (growth arrest and DNA damage inducible protein 45 alpha) to the rDNA promoter by TAF12, a TBP-associated factor that is contained in Pol I- and Pol II-specific TBP-TAF complexes. Once targeted to rDNA, Gadd45a triggers demethylation of promoter-proximal DNA by recruiting the nucleotide excision repair (NER) machinery to remove methylated cytosines. Knockdown of Gadd45a, XPA, XPG, XPF, or TAF12 or treatment with drugs that inhibit NER causes hypermethylation of rDNA, establishes heterochromatic histone marks, and impairs transcription. The results reveal a mechanism that recruits the DNA repair machinery to the promoter of active genes, keeping them in a hypomethylated state.

GTF2IRD1 regulates transcription by binding an evolutionarily conserved DNA motif ‘GUCE’

GTF2IRD1 is a member of a family of transcription factors whose defining characteristic is varying numbers of a helix–loop–helix like motif, the I-repeat. Here, we present functional analysis of human GTF2IRD1 in regulation of three genes ( HOXC8, GOOSECOID and TROPONIN I SLOW ). We define a regulatory motif (GUCE–GTF2IRD1 Upstream Control Element) common to all three genes. GUCE is bound in vitro by domain I-4 of GTF2IRD1 and mediates transcriptional regulation by GTF2IRD1 in vivo. Definition of this site will assist in identification of other downstream targets of GTF2IRD1 and elucidation of its role in the human developmental disorder Williams–Beuren syndrome.

Distinct modes of TATA box utilization by the RNA polymerase III transcription machineries from budding yeast and higher plants

The TATA box is a key upstream control element for basal tRNA gene transcription by RNA polymerase III in some eukaryotes, such as the fission yeast ( Schizosaccharomyces pombe) and higher plants, but not in others such as the budding yeast ( Saccharomyces cerevisiae). To gain information on this differential TATA box requirement, we examined side-by-side the in vitro transcription properties of TATA-containing and TATA-mutated plant and S. cerevisiae tDNAs in homologous in vitro transcription systems from both organisms and in a hybrid system in which yeast TBP was replaced by its plant homologue. The data support the general conclusion that specific features of the plant transcription machinery, rather than upstream region architecture per se, are responsible for the much stronger TATA box dependence of the plant system. In both systems, however, a strong influence of the TATA box on transcription start site selection was observed. This was particularly striking in the case of plant tDNAs, where TATA-rich upstream regions were found to favour the use of alternative initiation sites. Replacement of yeast TBP with its plant counterpart did not confer any general TATA box responsiveness to the yeast transcription machinery. Interactions involving components other than TBP are thus responsible for the strong TATA box requirement of plant tDNA transcription.

Epigenetic Mechanism of rRNA Gene Silencing: Temporal Order of NoRC-Mediated Histone Modification, Chromatin Remodeling, and DNA Methylation

Epigenetic control mechanisms silence about half of the rRNA genes in eukaryotes. Previous studies have demonstrated that recruitment of NoRC, a SNF2h-containing remodeling complex, silences rRNA gene transcription. NoRC mediates histone H4 deacetylation, histone H3-Lys9 dimethylation, and de novo DNA methylation, thus establishing heterochromatic features at the rRNA gene promoter. Here we show that inhibition of any of these activities alleviates NoRC-dependent silencing, indicating that these processes are intimately linked. We have studied the temporal order of epigenetic events at the rRNA gene promoter during gene silencing and demonstrate that recruitment of NoRC by TTF-I is a prerequisite for the deacetylation of histone H4 and the dimethylation of histone H3-Lys9. Inhibition of histone deacetylation prevents DNA methylation, while inhibition of DNA methylation does not affect histone modification. Importantly, ATP-dependent chromatin remodeling is required for methylation of a specific CpG dinucleotide within the upstream control element of the rRNA gene promoter, and this modification impairs preinitiation complex formation. The results of this study reveal a clear hierarchy of epigenetic events that control de novo DNA methylation and lead to silencing of RNA genes.

Molecular Mechanisms Mediating Methylation-Dependent Silencing of Ribosomal Gene Transcription

Epigenetic control mechanisms silence about half of ribosomal RNA genes (rDNA) in metabolically active cells. In the mouse, 40% of rDNA repeats are methylated and can be activated by 5-azacytidine treatment. In exploring the effect of methylation on rDNA transcription, we found that methylation of a single CpG dinucleotide within the upstream control element of the rDNA promoter (at −133) abrogates rDNA transcription both in transfection experiments and in in vitro assays using chromatin templates. Chromatin immunoprecipitation assays demonstrate that methylation of the cytosine at −133 inhibits binding of the transcription factor UBF to nucleosomal rDNA, thereby preventing initiation complex formation. Thus, methylation may be a mechanism to inactivate rDNA genes and propagate transcriptional silencing through cell division.

Basonuclin is associated with the ribosomal RNA genes on human keratinocyte mitotic chromosomes.

Basonuclin is a zinc finger protein mainly expressed in keratinocytes of the basal layer of epidermis and the outer root sheath of hair follicles. It is also found in abundance in the germ cells of testis and ovary. In cultured keratinocytes, basonuclin is associated with chromatin in all phases of the cell cycle, including mitosis. By immunocytochemical methods, we demonstrate here that in mitosis basonuclin is associated with the short arms of the acrocentric chromosomes and with other loci on many metaphase chromosomes of human keratinocytes. Using the evolutionarily highly conserved N-terminal pair of zinc fingers in an electrophoresis mobility shift assay, we demonstrate that the DNA target sequences of basonuclin on the acrocentric chromosomes are likely to be within the promoter region of the 45S rRNA gene transcription unit. DNase I footprinting shows that basonuclin zinc fingers interact with the upstream control element of this promoter, which is necessary for the high level of transcription of the rRNA genes. This result suggests that basonuclin may be a tissue-specific transcription factor for the ribosomal RNA genes.

Molecular structure and function of rat platelet-derived growth factor beta-receptor gene promoter.

To understand the regulatory mechanism of platelet-derived growth factor beta-receptor gene expression. A 1.7 kb genomic fragment was obtained from a rat genomic library. After we had determined an entire sequence of this fragment, transcription start sites were determined both by primer extension analysis and by riboprobe mapping. We performed a functional promoter assay by using a dual-luciferase reporter system. Progressive 5'-deletions of the fragment and site-directed mutagenesis for the CCAAT motif located at -67 or -94 were used for the assay, and their promoter activities in vascular smooth muscle cells were assessed. Gel-mobility shift analysis was also performed for the CCAAT motif at -67. Effects of the upstream sequence spanning -310 through -120 on heterologous gene promoters were also investigated. Multiple transcription start sites were observed in the 5'-flanking region, and the 1.7 kb sequence was actually active as a functional promoter in vascular smooth muscle cells. Two important sequences responsible for the basal transcriptional activity were identified by the functional promoter assay. One was the CCAAT motif at -67 which acts as a promoter itself, and the other was the upstream region spanning -310 through -210 which positively regulates the basal promoter activity. The basal promoter activity of the rat platelet-derived growth factor beta-receptor gene is mainly regulated by the interaction or coordination of two sequences, the CCAAT motif and the upstream control element.

Molecular Structure and Function of Rat CCAAT-Enhancer Binding Protein-Delta Gene Promoter

CCAAT-enhancer binding protein-delta (C/EBPδ) is a transcriptional nuclear factor, and belongs to basic region-leucine zipper class DNA binding proteins. One genomic clone containing a 12-kb sequence of the C/EBPδ gene was isolated from a rat genomic library, and a 2,056-bp fragment containing the 5′-flanking region was characterized. Sequence analysis of this fragment revealed that there were a TATA-like sequence (TAGAAAA) and many transcriptional regulatory elements. The transcription start site of the gene was determined by both primer extension analysis and riboprobe mapping. Both analyses indicated that the transcription start site was located at 31-bp downstream of the TATA-like sequence. Transient transfection experiments showed that the fragment cloned in this study was able to act as a functional promoter in rat vascular smooth muscle cells. The 5′-deletion analysis of this fragment revealed that the sequence spanning −235 through −82, which was designated as an upstream control element (UCE), remarkably increased a basal promoter activity of the C/EBPδ gene, and was also able to act as a promoter by itself. In addition, we also studied effects of the UCE on the heterologous gene promoter including rat α-actin gene promoter or SV40 virus promoter. Interestingly, the UCE specifically increased the promoter activity of the rat α-actin gene suggesting that the C/EBPδ gene may be positively controlled by the UCE via a cell-type or promoter-type specific manner.

A detailed mutational analysis of the VSG gene expression site promoter

The African trypanosome Trypanosoma brucei is a protozoan parasite that causes the disease African sleeping sickness. This parasite avoids the host's immune response by the process of antigenic variation, or by sequentially expressing antigenically different cell-surface coat proteins. These proteins, called variant surface glycoproteins (VSGs), are expressed from a specific locus, the VSG gene expression site (ES). In an attempt to understand expression of VSG genes, we expanded on earlier investigations of the promoter that controls the large VSG gene expression site transcription unit. We studied VSG ES promoter function both in transient transfection assays, and after stable integration at a chromosomal locus. Analysis of closely spaced deletion mutants showed that the minimum VSG ES promoter fragment that gives full activity is extremely small, and mapped precisely to a fragment that contains no more than −67 bp 5′ to the putative transcription initiation site. The promoter lacked an upstream control element, or UCE, an element found at the PARP promoter, and at most eukaryotic Pol I promoters. Furthermore, linker scanning mutagenesis demonstrated that the VSG ES promoter contains at least two essential regulatory elements, including sequences within the region −67 −60 and the region −35 −20 , both numbered relative to the initiation site. An altered promoter with mutated nucleotides surrounding the transcription initiation site still directed wild-type levels of expression. In this study, the results were similar for both insect and bloodstream form trypanosomes, suggesting that the same basic machinery for expression from the VSG ES promoter is found in both stages of the parasite.

The RNA polymerase I transcription factor, upstream binding factor, interacts directly with the TATA box-binding protein.

The accurate transcription of human rRNA genes by RNA polymerase I requires two transcription factors, upstream binding factor (UBF) and promoter selectivity factor (SL1). Human SL1 (hSL1) is a multisubunit complex, one of whose components is TATA box-binding protein (TBP). hSL1 binds to the core region of the rRNA promoter, but does so inefficiently in the absence of human UBF (hUBF). hUBF interacts with the upstream control element of the rRNA promoter and facilitates binding of hSL1. The molecular basis by which hUBF increases binding of hSL1 remains to be elucidated. In this report, we use an immobilized protein binding assay to identify and purify a 95-kDa TBP-binding polypeptide. Microsequence analysis reveals that the 95-kDa TBP-binding protein is hUBF. We show that hUBF is stably associated with TBP and is present in large TBP-containing complexes. Our results indicate that the cooperative binding of hUBF and hSL1 on the rRNA promoter is mediated by direct interaction between hUBF and TBP. We also provide evidence that hUBF associates with TFIID, a TBP-containing RNA polymerase II transcription factor.

Comparative analysis of the sequence and structure of two Drosophila melanogaster genes encoding vitelline membrane proteins

Two Drosophila melanogaster vitelline membrane protein-encoding genes ( VM), located at polytene band positions 26A and 34C, have been cloned and comparatively characterized at the nucleotide level. Sequence analysis of genomic and cDNA clones for the two genes, VM26A.1 and VM34C.1, indicates that both are similarly organized with a central highly conserved domain [Scherer et al., Dev. Biol. 130 (1988) 786–788] which is flanked by unrelated regions, and that both genes lack introns. Comparison of the upstream regions reveals that both VM genes contain a heptameric element identical to one associated with the D. melanogaster yolk protein-encoding genes ( YP). This heptamer occurs in the specific 5' flanking region responsible for ovarian temporal- and tissue-specific control in both VM and YP genes. A putative chorion transcription factor 2 site is also associated with an upstream control element of VM26A.1, but not with any sequenced portion of VM34C.1.