Duck lactate dehydrogenase B/epsilon-crystallin gene. Lens recruitment of a GC-promoter.

In duck the single copy lactate dehydrogenase B (LDH-B) gene also encodes an abundant lens protein, epsilon-crystallin. The LDH-B/epsilon-crystallin gene consists of eight exons, of which the first is non-coding. The promoter region lacks a TATA box, is very GC-rich and contains multiple consensus Sp1 binding sites. The gene has two discrete transcription start sites located 28 base-pairs apart. Both sites are used about equally in heart tissue, while transcription from the downstream start site predominates in the lens. For maximal promoter activity in lens or heart, sequences from the first intron are required. The enhancer(s) in this intron is promoter specific as it could not activate the tk promoter. Studies at the RNA level show that the overexpression of the LDH-B/epsilon-crystallin gene in the lens is regulated at the transcriptional level, yet no tissue-specific regulatory elements could be detected in a region spanning from -1.9 kb (1 kb = 10(3) bases or base-pairs) up to the translation initiation site in the second exon. The basis for the differential expression of the LDH-B/epsilon-crystallin gene in duck heart and lens is the usage of the downstream transcription initiation site. However, our results do not allow a distinction between activation of the downstream transcription start site in the lens or repression of the use of this site in heart.

The mechanism of recruitment of the lactate dehydrogenase-B/epsilon-crystallin gene by the duck lens.

In duck, the housekeeping enzyme lactate dehydrogenase B (LDH-B) and the lens structural protein epsilon-crystallin are encoded by the same single copy gene. Transcription of the gene is initiated from two closely spaced start sites, at -28 and +1. The usage of the downstream site is greatly enhanced in lens. Deletion mapping of the promoter shows that the region -70/+18 specifies the enhanced promoter activity in the lens. A critical role is played by the consensus Sp1 binding site at -50; mutation of this site abolishes lens-preferred expression. Deletion of the +1 transcription initiation site also leads to a decrease in lens-preferred expression, which can be restored by moving the -28 transcription initiation site downstream. By band shift experiments, supershift mobility assays and methylation interference assays, Sp1 was shown to bind to the Sp1 consensus binding site at -50 using either heart or lens nuclear extracts. Co-expression of Sp1 or Sp1-like factors inhibited the activity of an LDH-B/epsilon-crystallin promoter construct by approximately 60% in lens and by 40% in heart cells. Co-expression of Pax-6, a transcription factor shown to be involved in the lens-enhanced expression of a number of other crystallin genes, did not influence the promoter activity of the -130/+650 LDH-B/epsilon-crystallin promoter construct. In contrast to other crystallin promoters, the LDH-B/epsilon-crystallin promoter does not appear to contain a lens-specific element, rather our data lead to a model in which a factor transmitting the effect of Sp1, bound at -50, to the transcription initiation complex is responsible for the lens-preferred expression of the LDH-B/epsilon-crystallin promoter.

Activation and repression sequences determine the lens-specific expression of the rat gamma D-crystallin gene.

Rat lens nuclear extracts contain a factor that binds to position -57 to -46 of the rat gamma D-crystallin promoter region. This factor protects the sequence 5'-CTGCCAACGCAG-3' in a footprint analysis. Binding to this region is crucial for maximal promoter activity in rat lens cells, but this sequence was unable to act as an enhancer when cloned in front of a heterologous promoter. A region directly upstream from this activating sequence, between position -85 to -67, acts as a strong silencer of promoter activity in non-lens cells. This silencing effect is mediated by trans-acting factor(s). Our data provide evidence for two regulatory elements in rat gamma D-crystallin gene expression, an activating sequence active in lens cells and a silencing sequence active only in non-lens cells. The factor that binds to the activating sequence could be detected only in lens cells and may be a determinant of the lens-specific expression of the gamma-crystallin genes.

The developmental expression of taxon-specific crystallins in the duck lens.

The three crystallins, alpha B-crystallin, tau-crystallin/alpha-enolase and epsilon-crystallin/lactate dehydrogenase B are all stress induced proteins and may thus share common regulatory elements. However, no evidence was found for coordinate expression of these three genes during duck lens development. The alpha B- and alpha-enolase/tau-crystallin mRNAs accumulate with similar kinetics between day 12 and day 24 of embryonic development but differ in their epithelial versus fibre cell location; the LDH-B/epsilon-crystallin transcript shares its preferential location in the fibre cell with alpha B-crystallin but differs in its developmental pattern of expression. The accumulation of LDH-B/epsilon-crystallin mRNA in heart and lens followed a similar developmental pattern. In contrast, the alpha-enolase/tau-crystallin mRNA level in heart decreases while the level in lens rises. The LDH-B gene is used as a crystallin gene in duck but not in chicken. This species-specific difference may correlate with the difference in LDH-B activity between various chicken and duck tissues: the retina and pancreas in duck have significantly higher LDH-B activity than in chick, while heart, muscle, stomach, liver, intestine and kidney all have much higher LDH-B activity in chicken than in duck.

Stimulation of the methylcoenzyme M reduction by uridine-5'-diphospho-sugars in cell-free extracts of Methanobacterium thermoautotrophicum (strain delta H).

The hydrogen-dependent reduction of methylcoenzyme M catalyzed by coenzyme-depleted cell-free extracts of Methanobacterium thermoautotrophicum was stimulated by micromolar concentrations of a UDP-disaccharide present in the organism. The compound was isolated and identified as UDP-1-O-alpha-D-2-acetamido-2-deoxyglucopyranose (UDPGlcpNAc) glycosidically linked to 2-acetamido-2-deoxymannopyranosyluronic acid. Maximal stimulation was observed when both the UDP-disaccharide and mercaptoheptanoylthreonine phosphate were present in the reaction mixtures. The UDP derivative isolated was not specific in its action: other UDP-sugars tested in micromolar concentrations stimulated the methylcoenzyme M reduction to the same extent. The activated sugars presumably substitute for ATP, which is usually required in much higher concentrations to activate the methylcoenzyme M reductase system.

Aberrant platelet-derived growth factor alpha-receptor transcript as a diagnostic marker for early human germ cell tumors of the adult testis.

Testicular germ cell tumors are the most common form of cancer in young adult males. They result from a derangement of primordial germ cells, and they grow out from a noninvasive carcinoma-in-situ precursor. Since carcinoma in situ can readily be cured by low-dose irradiation, there is a great incentive for non- or minimally invasive methods for detection of carcinoma in situ. We have recently shown that human Tera-2 embryonal carcinoma cells, obtained from a nonseminomatous testicular germ cell tumor, show alternative splicing and alternative promoter use of the platelet-derived growth factor alpha-receptor gene, giving rise to a unique 1.5-kb transcript. In this study we have set up a reverse transcriptase-polymerase chain reaction strategy for characterization of the various transcripts for this receptor. Using this technique, we show that a panel of 18 seminomas and II nonseminomatous testicular germ cell tumors all express the 1.5-kb transcript. In addition, a panel of 27 samples of testis parenchyma with established carcinoma in situ were all found to be positive for the 1.5-kb transcript, while parenchyma lacking carcinoma in situ, placenta, and control semen were all negative. These data show that the 1.5-kb platelet-derived growth factor alpha-receptor transcript can be used as a highly selective marker for detection of early stages of human testicular germ cell tumors.

The cooperation between two silencers creates an enhancer element that controls both the lens-preferred and the differentiation stage-specific expression of the rat beta B2-crystallin gene.

The rat beta B2-crystallin gene is active only during a specific stage of the differentiation of rat lens fibre cells directed by basic fibroblast growth factor. The regulatory elements that determine the transient activity of this gene are located in the -750/-123 region and in the first intron. Singly, these elements act as silencers, together they constitute an enhancer that is active only during the specific differentiation stage. An additional silencer is found between -123 and -77. The proximal promoter region contains a Pax-6 binding site at -65/-51. In vitro, binding to this site could be detected but, according to in vivo footprinting experiments, this site is not occupied in the endogenous gene. Furthermore, co-expression of Pax-6 did not enhance promoter activity. Finally, mutation or deletion of this site did not affect promoter activity: the region -37/+10 sufficed for basal promoter activity. The cooperation between the -750/ -123 region and the first intron of the beta B2-crystallin gene not only determines the differentiation stage-specific activity of the gene, but also contributes to the highly increased expression in lens cells compared with non-lens cells.

Oct-4 regulates alternative platelet-derived growth factor alpha receptor gene promoter in human embryonal carcinoma cells.

Expression of the platelet-derived growth factor alpha-receptor (PDGFalphaR) gene is tightly controlled in mammalian embryogenesis. A well established model system to study human embryogenesis is the embryonal carcinoma cell line Tera2. We have shown previously that retinoic acid-differentiated Tera2 cells express two PDGFalphaR transcripts of 6.4 kilobase pairs (kb) (encoding the full-length receptor) and 3.0 kb, respectively, whereas in contrast, undifferentiated Tera2 cells express PDFGalphaR transcripts of 1.5 kb and 5.0 kb. Here we show that this switch in PDGFalphaR expression pattern during differentiation of Tera2 cells results from alternative promoter use. In undifferentiated cells, a second promoter is used, which is located in intron 12 of the PDGFalphaR gene. Functional analysis shows that this promoter contains a consensus octamer motif, which can be bound by the POU domain transcription factor Oct-4. Oct-4 is expressed in undifferentiated Tera2 cells but not in retinoic acid-induced differentiated cells. Mutation of the octamer motif decreases promoter activity, while ectopic expression of Oct-4 in differentiated Tera2 cells specifically enhances the activity of this PDGFalphaR promoter. Therefore, we suggest that an important aspect in the maintenance of the undifferentiated state of human embryonal carcinoma cells results from Oct-4 expression, which thereupon activates this PDGFalphaR promoter.