Genes for human general transcription initiation factors TFIIIB, TFIIIB-associated proteins, TFIIIC2 and PTF/SNAPC: functional and positional candidates for tumour predisposition or inherited genetic diseases?

TFIIIB, TFIIIC2, and PTF/SNAPC are heteromultimeric general transcription factors (GTFs) needed for expression of genes encoding small cytoplasmic (scRNAs) and small nuclear RNAs (snRNAs). Their activity is stimulated by viral oncogenes, such as SV40 large T antigen and Adenovirus E1A, and is repressed by specific transcription factors (STFs) acting as anti-oncogenes, such as p53 and pRb. GTFs role as final targets of critical signal transduction pathways, that control cell proliferation and differentiation, and their involvement in gene expression regulation suggest that the genes encoding them are potential proto-oncogenes or anti-oncogenes or may be otherwise involved in the pathogenesis of inherited genetic diseases. To test our hypothesis through the positional candidate gene approach, we have determined the physical localization in the human genome of the 11 genes, encoding the subunits of these GTFs, and of three genes for proteins associated with TFIIIB (GTF3BAPs). Our data, obtained by chromosomal in situ hybridization, radiation hybrids and somatic cell hybrids analysis, demonstrate that these genes are present in the human genome as single copy sequences and that some cluster to the same cytogenetic band, alone or in combination with class II GTFs. Intriguingly, some of them are localized within chromosomal regions where recurrent, cytogenetically detectable mutations are seen in specific neoplasias, such as neuroblastoma, uterine leyomioma, mucoepidermoid carcinoma of the salivary glands and hemangiopericytoma, or where mutations causing inherited genetic diseases map, such as Peutz-Jeghers syndrome. Their molecular function and genomic position make these GTF genes interesting candidates for causal involvement in oncogenesis or in the pathogenesis of inherited genetic diseases.

Melanosynthesis, differentiation, and apoptosis in Kupffer cells from Rana esculenta.

It is well known that amphibian Kupffer cells (KCs) contain eumelanins. In this paper, we demonstrate through a molecular analysis that Rana esculenta KCs synthesize high levels of mRNA for tyrosinase and through cytochemistry that they possess dopa oxidase activity: both these data prove that frog KCs are capable of autonomously synthesizing eumelanins. On the other hand, by using a highly sensitive reverse transcription-polymerase chain reaction assay we clearly show that in mammalian KCs the tyrosinase gene is not expressed. Quite unexpectedly, we have detected tyrosinase mRNA in Rana esculenta spleen, lung, and heart; to explain this finding, we suggest that it could be due to the presence of pigmented macrophages within the spleen, that probably behave as KCs, and of melanophores in lung and heart. It also may be hypothesized that the Rana esculenta tyrosinase gene, as opposed to its mammalian counterpart, is expressed in many cell types because its promoter contains sequences that are recognized by widely synthesized transcription factors. Our experiments also demonstrate that there is an inverse correlation between the amount of tyrosinase mRNA and melanin content, and that populations of terminally differentiated KCs are characterized by a high degree of apoptosis. Based on these data, we propose that differentiating KCs start accumulating eumelanins, as a result of previous expression of high levels of tyrosinase and of dopa oxidase activity, acquire the full KC phenotype (characterized by both phagocytic and melanosynthetic ability), and then undergo apoptosis. Accordingly, we propose that these cells could represent an interesting model to study, at the molecular level, the relationship between differentiation, specific gene expression, and programmed cell death in higher eukaryotes.

Genomic localization of the human genes TAF1A, TAF1B and TAF1C, encoding TAF(I)48, TAF(I)63 and TAF(I)110 subunits of class I general transcription initiation factor SL1.

Human SL1 is a general transcription initiation factor (GTF) essential for RNA polymerase I to start rRNA synthesis at class I promoters. It is comprised of the TATA box-binding protein (TBP) and three TBP-associated factors (TAF(I)48, TAF(I)63 and TAF(I)110). We have determined that the human genes TAF1A, TAF1B and TAF1C, encoding these three TAF(I) polypeptides, are localized at lq42, 2p25 and 16q24, respectively. All three genes are present as single copies in the human genome and map to different chromosomes, as shown by somatic cell hybrid panel and radiation hybrid panel analysis and FISH. Two of these genes, TAF1C and TAF1B, are transcribed into multiple RNAs, as determined through Northern analysis of mRNA from various human organs and cell lines. If translated into different polypeptides, this could result in production of variant isoforms of SL1 with different activation potentials.

Characterisation of Kupffer cells in some Amphibia.

A study on the Kupffer cells (KCs) of Amphibia was undertaken in order to compare these cells with those of endothermic animals. Liver tissue and isolated and cultured KCs were studied by light microscopy and by transmission and scanning electron microscopy. We have shown that amphibian KCs can be divided into 2 principal types: 'small' and 'large'. Both cell types possess the distinctive KC morphology. They show nonspecific esterase activity, weak endogenous peroxidase activity in the nuclear envelope and in the rough endoplasmic reticulum, and the ability to engulf naturally present cell debris or experimentally administered zymosan or latex particles. The principal difference between the small and the large cells consists in the substantial quantity of inclusion bodies that exist only in the latter. We conclude that amphibian KCs, apart from their ability to build melanosomes and synthesise melanins, are very similar to mammalian KCs.

Genomics of the human genes encoding four TAFII subunits of TFIID, the three subunits of TFIIA, as well as CDK8 and SURB7.

By in situ chromosomal hybridization, and by somatic cell and radiation hybrid analysis, we have determined the genomic position of the human genes encoding four TAFII subunits of TFIID (TAFII150, TAFII105, TAFII68, TAFII18), the three subunits of TFIIA (TFIIA35 and TFIIA19, both encoded by the same gene, and TFIIA12), CDK8, and SURB7. All of these proteins are bona fide components of human class II holoenzymes as well as targets of signal transduction pathways that regulate genome expression. The genes encoding them are present in the human genome in a single copy and are localized at 8q23, 18q11.2, 17q11.1-11.2, 1p21, 14q31, 15q21-23, 13q12, and 12p12, respectively. We have mapped all of them to chromosomal regions where hereditary genetic diseases have been localized or which are involved in malignancies, which makes them potential candidates for a causal involvement in these phenotypes.

Genomics and transcription analysis of human TFIID.

TFIID, a multisubunit protein comprised of TBP (TATA box-binding protein) and TAF(II)s (TBP-associated factors), has a central role in transcription initiation at class II promoters. TAF(II)s role as mediators of regulatory transcription factors, such as pRb and p53, and their involvement in signal transduction pathways suggest that some may participate in the control of cell proliferation and differentiation: therefore, they could be considered potential protooncogenes or antioncogenes. With the aim of starting to analyse these potential roles, we have determined the genomic position of nine human TAF(II) genes (TAF[II]250, TAF[II]135, TAF[II]100, TAF[II]80, TAF[II]55, TAF[II]43, TAF[II]31, TAF[II]28, TAF[II]20/15) and of two previously unknown sequences related to TAF(II)250 and TAF(II)31, respectively. Except for those encoding TAF(II)250 and TAF(II)31, these genes are present in a single copy and, with the exclusion of those for TAF(II)43 and TAF(II)28 (both at 6p21), are localized in different segments of the genome. Indeed, six of them map to a chromosomal region commonly altered in specific neoplasias, which defines them as candidates for involvement in oncogenesis. Our experiments also demonstrate that TAF(II) transcripts are synthesized ubiquitously, mostly at low levels similar to those of TBP. Interestingly, the amount of the major mRNA species detected by TAF(II)20/15 cDNA is higher, which suggests that the polypeptide it encodes may also perform functions independently of TFIID. TAF(II) isoforms, indicated by additional bands on Northern blots, may play a role in modulation of TFIID function. These data will be useful for analysing variations of TAF(II) mRNA phenotype during cell proliferation, differentiation and development, both normal and pathological.

The amphibian Kupffer cells build and demolish melanosomes: an ultrastructural point of view.

This ultrastructural research was carried out to investigate the nature of the liver pigment cells of anuran and caudate amphibians, the pattern of melanosome ontogenesis, and the demolition processes of old melanosomes. We demonstrate that these liver pigment cells are able to internalize zymosan particles and latex beads; therefore, being professional phagocytes, they, as liver resident macrophages, can be classified as Kupffer cells (KCs). They show "melanosomogenesis centers" in which several maturation stages of premelanosomes are visible; the premelanosomes are formed by two principal components: a filamentous structure that will constitute the "inner" area of the melanosome and a vesicular component, budding from the Trans Golgi Network and that carries enzymes, which will constitute the "cortical area" of the melanosome. Thus the KCs, thanks to the presence of the "melanosomogenesis centers," are also melanosome producing cells. They are also able to demolish melanosomes by heterophagocytosis and, probably, also by autophagocytosis. In conclusion, we propose a classification of vertebrate pigment cells.

Study of a melanic pigment of Proteus mirabilis.

The present study sought to determine whether the pigment produced by Proteus mirabilis from the L-forms of various aromatic amino acids under aerobic conditions is melanic in nature. It is a black-brown pigment which behaves like a melanin in many respects, namely solubility features, bleaching by oxidizing agents and positive response to the Fontana-Masson assay. In the present study, for the first time, it was shown by electron spin resonance analysis that a bacterial melanin is able to act as a free radical trap, as was previously demonstrated for other melanins. Scanning electron microscopy studies showed a specific organized structure of the pigment as rounded aggregates of spherical bodies. DNA hybridization data did not reveal, in the P. mirabilis genome, any nucleotide sequence related to Shewanella colwelliana mel A, one of the two melanogenesis systems already defined at the molecular level in bacteria. Results obtained from experiments on pigment production inhibition suggest a possible role of tyrosinase in P. mirabilis melanogenesis. In conclusion, from the bulk of our results, it appears that the pigment produced by P. mirabilis is melanic in nature.

Genomic localization of the human gene encoding Dr1, a negative modulator of transcription of class II and class III genes.

Dr1 is a nuclear protein of 19 kDa that exists in the nucleoplasm as a homotetramer. By binding to TBP (the DNA-binding subunit of TFIID, and also a subunit of SL1 and TFIIIB), the protein blocks class II and class III preinitiation complex assembly, thus repressing the activity of the corresponding promoters. Since transcription of class I genes is unaffected by Dr1. it has been proposed that the protein may coordinate the expression of class I, class II and class III genes. By somatic cell genetics and fluorescence in situ hybridization, we have localized the gene (DR1), present in the genome of higher eukaryotes as a single copy, to human chromosome region 1p21-->p13. The nucleotide sequence conservation of the coding segment of the gene, as determined by Noah's ark blot analysis, and its ubiquitous transcription suggest that Dr1 has an important biological role, which could be related to the negative control of cell proliferation.

Melanins in physiological conditions protect against lipoperoxidation. A study on albino and pigmented Xenopus.

The susceptibility to lipoperoxidation in liver of albino and pigmented Xenopus laevis Daudin, has been studied. Albino Xenopus liver was richer in polyunsaturated fatty acids (PUFAs) than the pigmented one; moreover, it was also richer in mitochondrial superoxide dismutase (MnSOD) and in reduced glutathione (GSH). The thiobarbituric acid-reactive substances (TBARS) values were more abundant in the albino tissue compared to the pigmented tissue both during spontaneous and Fe++ induced lipoperoxidation. Therefore, when isolated and purified melanin, in physiological quantities, was added to albino tissue, the TBARS values drastically decreased. Thus, melanin, in our experimental conditions, protects the albino tissue even more than SOD and GSH do. Melanin, in our opinion, acts as an antioxidant, because it is able to scavenge O2-.

Genetic characterization of general transcription factors TFIIF and TFIIB of Homo sapiens sapiens.

Analysis of loci GTF2F1 and GTF2B, encoding Rap 74 (a subunit of TFIIF) and TFIIB, respectively, showed that they are present in a single copy in the human genome and are localized at 19p13.3 and 1p22, respectively. By using as probe a cDNA for Rap 30 (the other subunit of TFIIF), we localized the GTF2F2 locus to 13q14; the same probe also detected a cross-hybridizing sequence at 4q31 whose functional importance remains to be elucidated. These data and those previously published by our group demonstrate that genes coding for class II general transcription factors with reported sequence similarity to bacterial sigma proteins are scattered in different regions of the human genome, with no evidence of clustering. This dispersion and the identification of homologs of both TBP and TFIIB in Archaea suggest an early evolutionary origin of the general transcription apparatus of contemporary eukaryotes.

Localization of the human genes encoding the two subunits of general transcription factor TFIIE.

TFIIE is a general transcription factor for class II genes composed of two types of subunits, a large one of 56 kDa and a small of 34 kDa. By Southern analysis at high and at low stringency of a panel of mouse/human hybrid cell lines and by in situ chromosomal hybridization, we have demonstrated that both polypeptides are encoded by genes that are single copy in the human genome and are localized at 3q13-q21 and at 8p12, respectively. A TaqI RFLP (heterozygosity index of 0.07) was detected at the locus for the 56-kDa subunit.

ATPase activity of melanosomes in liver pigment cells of Rana esculenta L.

Luminometric methods show that melanosomes in liver pigment cells of Rana esculenta L. have endogenous ATP and ATPase activity. The Km value of ATPase is 0.42 x 10(-8) mol/l at pH 7.0. Inhibition of ATPase by antimycin and by ouabain is not effective. In the presence of an excess of ADP and Pi, ATP synthesis was observed.

Physical mapping at 6q27 of the locus for the TATA box-binding protein, the DNA-binding subunit of TFIID and a component of SL1 and TFIIIB, strongly suggests that it is single copy in the human genome.

The TATA box-binding protein (TBP) has a fundamental role in eukaryotic cell metabolism, since it is necessary for transcription of class I, class II, and class III genes; in fact, TBP is the DNA-binding subunit of TFIID and a component of SL1 and TFIIIB. Contrary to the previously hypothesized existence of a family of genes coding for DNA-binding proteins highly related to TBP, our experiments show that the segment coding for the evolutionarily well-conserved carboxyl-terminal domain, involved in DNA binding, is unique; accordingly, we conclude that the TBP locus itself, which we have localized to 6q27, is single copy in the human genome. On the other hand, a cDNA fragment coding for the evolutionarily variable amino-terminal domain detects multiple cross-hybridizing sequences in the genome of higher eukaryotes. We suggest that the common motif is represented by the long string of glutamine codons, which characterizes the amino-terminal segment of human TBP: in fact, other proteins involved in transcription, such as TAF II 110, Sp1, and some homeobox proteins, are known to contain glutamine-rich segments.

The gene for SP-40,40, human homolog of rat sulfated glycoprotein 2, rat clusterin, and rat testosterone-repressed prostate message 2, maps to chromosome 8.

Sulfated glycoprotein 2 (SGP-2) is a rat glycoprotein that is particularly abundant in seminal fluid, where it is found associated with the acrosome and the tail of mature spermatozoa; for this reason it has been suggested that it has an important role in spermatogenesis. On the basis of nucleotide sequence homology, it has been proposed that the orthologous human gene is that coding for serum protein-40,40 (SP-40,40), a serum protein also called complement lysis inhibitor (CLI), SP-40,40 has been shown to act as a control mechanism of the complement cascade: in fact, it prevents the binding of a C5b-C7 complex to the membrane of the target cell and in this way inhibits complement-mediated cytolysis. SGP-2 and SP-40,40 seem then to be part of different biological systems. Furthermore it has been shown that another protein, testosterone-repressed prostate message 2 (TRPM-2), shares sequence homology with SGP-2 and SP-40,40. TRPM-2 is expressed at high levels and in a temporally precisely defined manner in dying cells, an observation that would suggest its involvement in the cascade of events leading to cell death. We have used a large panel of 24 mouse/human hybrid cell lines and a cDNA for SGP-2, which is also highly homologous to that for rat clusterin, to map the chromosomal location of the orthologous human gene. The mapping data and the Southern analysis presented in this paper, in addition to the data available from the literature, strongly suggest that in the human genome there is a single locus homologous to the probe used and that it codes for the protein which has been called, in different species, SP-40,40, SGP-2, clusterin, and TRPM-2. The chromosomal mapping of the locus for this multiname protein should facilitate its cloning and a better understanding of the apparently many biological functions of its product.

In vitro scavenger activity of some flavonoids and melanins against O2-(.).

The scavenger activity against O2-. of some flavonoids and melanins (synthetic melanins and melanins isolated from animal tissues, vegetable seeds, and mushroom spores) has been studied by ESR spectrometry. All these substances, except flavon and flavanone, diminish the signal of O2-. generated in vitro by a system containing H2O2 and acetone in an alkaline medium. It is shown that the presence of hydroxyl groups in the B ring of flavonoids is essential for their scavenger activity. Moreover, the presence of a hydroxyl at C-3 enhances the scavenger ability of flavonoids. Generally, aglycons are more active than their glycosides. It seems plausible that the antioxidant property of these substances comes from their scavenger activity against O2-(.). It is also pointed out that the scavenger activity shown by melanins, is strictly correlated with their nature of stable free radical.

Circannual rhythm of the melanin content in frog liver (Rana esculenta L.).

The melanin content of Rana esculenta L. liver varies according to a circannual statistically significant rhythm, as shown by variance and single cosinor analysis. The maximum is found in autumn-winter, the minimum in spring-summer. The linear regression analysis shown a negative correlation between the amount of melanin and the environmental temperature.

Lipid peroxidation in pigmented and unpigmented liver tissues: protective role of melanin.

The protective role of melanin as an antioxidant biopolymer against lipid peroxidation was investigated. In pigmented frog liver and in albino rat liver the following were tested: thiobarbituric acid (TBA) reactive material (to show the induced lipoperoxidation in vitro), fatty acids, and reduced glutathione content. Our results show that susceptibility to the in vitro lipoperoxidation induced by ferrous ions is lower in the tissue containing melanin, though the content of the polyunsaturated fatty acids is higher in pigmented than in unpigmented tissues and reduced glutathione levels are lower in pigmented tissue. Our data support the hypothesis that melanin could reduce lipoperoxidation in pigmented tissue.

Antioxidant enzymatic systems in pigment tissue of amphibia.

Superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities in pigmented and unpigmented liver tissues of frog and albino rat, respectively, were studied. Our results show that pigmented tissue is lacking in manganese superoxide dismutase activity and that the main enzymatic activity utilized in the cytosol by pigmented cells to reduce the hydrogen peroxide to water is represented by catalase; on the contrary, for the same reaction, the cells of albino rat liver primarily utilize the glutathione peroxidase activity. Both a low glutathione peroxidase activity and a low glutathione reductase activity were found in pigmented tissue of frog liver when compared with unpigmented tissue of rat liver. In light of our results, we also report a hypothetical interrelationship between melanin and reduced glutathione: We believe that in pigmented cells the melanin could act as a reducing physiological agent replacing the glutathione in the reduction of hydrogen peroxide. This reducing action of melanin could cause a diminished need for GSH and therefore could provoke the low glutathione peroxidase and reductase activities in pigmented tissue.

Biosynthesis and function of melanins in hepatic pigmentary system.

In this report we show the most important results obtained from our study of the pigment liver cells of Amphibia and Reptilia. Contrary to the cutaneous pigment cells that derive from the neural crest, the liver pigment cells, instead, derive from the Kupffer cells: this can be seen from the results obtained from labelled precursor incorporation experiments, not only in vivo but also in vitro, using surviving liver slices or isolated melanosomes. Chemical analysis of both liver and cutaneous melanosomes reveal a great difference in their chemical composition, and this is in agreement with the different origins of these cells. We therefore propose that: liver pigment cells of Amphibia and Reptilia should be classified as "Extra Cutaneous Pigment Cells from Histocytic Origin". As regards the function of melanins, we show that O2 is trapped by these substances. Moreover, in my laboratory we have shown in several animal species, that the superoxide dismutase activity is inversely proportional to the quantity of melanin present; thus, we think that melanin could mime SOD activity.