Resultados de los cuestionarios de las Normas de control

The fibrinogen gene locus has been mapped to the distal third of the long arm of human chromosome 4 (Olaisen et al 1982, Humphries et al 1984, Kant et al 1985). As

42

described in the preceeding section, the fibrinogen molecule consists of three polypeptide chains A a, and 7. This six chain a2-^2-y2 structure has been found to

be conserved in even the most primitive vertebrates, where 50% homology exists between the amino acid sequences of the |8 and 7 chains and those in the human genes

(Strong et al 1985, Bohonus et al 1986). Each polypeptide chain is encoded by a separate mRNA transcribed from three single-copy genes (Imam et al 1983, Kant et al 1983b, Kant et al 1985). Cloning of the cDNAs for each polypeptide chain and comparison of the cDNAs and the amino acid sequences showed a strong homology between these genes, suggesting they are evolutionarily related (Henschen and Me Donagh 1986). Further investigation showed that these three genes lie in close proximity to each other over a region of 50 kilobases (Kant et al 1985) and that the jS gene is in the opposite orientation to the a and 7 genes (Kant et al 1983b, Kant et al

1985). Kant suggested that gene duplication and inversion of a single ancestral gene resulted in the present organization of the fibrinogen genes, which have the same arrangements o f introns and exons (Kant et al 1985). The entire sequences of the fibrinogen a-, /?-, and 7-chain genes are known and are deposited in the Genbank

databank (Rixon et al 1983, Chung et al 1983, Rixon et al 1985).

The process of transcription of a DNA sequence into RNA is central to gene expression and is therefore one of the major points of control. Tissue-specific or signal-specific protein production has been shown to be dependent largely on transcriptional control (Darnell 1982). In eukaryotic genes, regions upstream of the sequence which encode the final protein have been found to contain two types of elements:- those found in many genes, which appear to be involved in the basic transcription process, and those found in genes whose transcription is tissue or signal specific. These sequences are

43

known as cw-acting elements, and the proteins which bind to such sequences are known as trans-2iC\mg focfors .

The TATA box is an AT-rich region (consensus TATAA/TAA/T) which is found about 30 base pairs upstream of the transcriptional start site in many genes. The TATA box is involved in binding RNA polymerase II, the enzyme responsible for transcribing protein coding genes, and is involved in the accurate positioning o f the start site of transcription (Goodwin et al 1990). The Spl box is a GC-rich sequence, one or more copies of which are found upstream of the promoter in many genes, that dramatically enhances the low activity of the promoter itself (Maniatis et al 1987). The CCA AT box is also found typically upstream of the TATA box and is present in many but not all genes. The importance of these elements is shown by the fact that the elimination of any of these sites by mutation or deletion results in the abolition of transcription (Me Knight et al 1984).

Sequences involved in regulated transcription are less frequent and are found interspersed with the elements described above. There are a variety of elements found in signal-specific genes: the heat shock consensus element is involved in the regulation of transcription in response to heat and is found in, among others, the human heat- shock protein (hsp 70) gene. Other signal-specific elements are the glucocorticoid response element which can bind the glucocorticoid hormone-receptor complex, the metal response elements (MRE) which allow activation of the gene in response to treatment with heavy metals such as zinc and cadmium and elements which bind a transcription factor AP2 which allows activation of the gene in response to cyclic AMP and phorbol esters. Other similar elements are found in the promoters o f tissue-specific

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genes, but these bind transcription factors which are active only in tissue where the gene is expressed. These include the octamer motif in the ^ h a n e e r s of the immunoglobulin heavy- and light- chain genes which mediates B cell-specific expression by binding the transcription factor Oct-2 and a sequence in the promoters of the prolactin and growth hormone genes which mediates expression only in pituitary cells (Reviewed by Latchmanl991).

The fibrinogen genes each contain elements involved in the basic transcriptional process and elements involved in tissue- and signal-specific transcription. As previously descibed, formation of the /3-fibrinogen chain is the rate limiting step in the biosynthesis o f fibrinogen and therefore the control of the expression of the jS-fibrinogen gene may be important in determining plasma levels of fibrinogen (Yu et al 1983, 1986).

All the human fibrinogen genes have a TATA box and a CAAT box (Courtois et al 1987, Morgan et al 1988). In the jS gene there is a TATA box at -21 to -29 and a CAAT box at -58 to -63 (Huber et al 1987). The TATA box of the y-gene was found to be different and closer to the transcription start point although the CAAT boxes of both genes are identical and at approximately the same distance from the transcripiton start site (Rixon et al 1985). There are also homologous sequences present in the ot- fibrinogen gene (Rixon et al 1985, Huber et al 1987). The j8 fibrinogen gene has both

distal and proximal elements which regulate its expression in response to cytokines and hormones.

Studies on the fibrinogen gene promoter showed that it was functional in hepatoma cells but not in non-hepatic cell lines, and this correlated with the presence or absence

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o f a liver-specific nuclear factor, hepatic nuclear factor (HNF-1) (Courtois et al 1987). This factor was found to interact with a short sequence between -89 and -77 upstream of the /S fibrinogen gene (Courtois et al 1987). The HNF-1 binding site was also found to be present in the a-fibrinogen promoter and in the promoters of other liver-specific genes (Courtois et al 1988, Kugler et al 1988, Costa et al 1989, Baumhueter et al

1990).

The control of the 7-fibrinogen gene appears to be different from that of the a - and /3-

genes and the cis- and trans-zciing factors which have been found in the promoter of this gene are not hepatic-specific. Binding sites for a CAAT-binding protein, an Spl transcription factor and the adenovirus major late transcription factor were found (Chodosh et al 1987, Morgan et al 1988). Since none of these factors are liver-specific, there may still be undiscovered elements which are involved in the tissue specific expression of this gene, or it has been suggested that perhaps the H N Fl element of the a-fibrinogen gene may act as an enhancer to control the expression of the 7-fibrinogen

gene as well as the a-fibrinogen gene (Green and Humphries 1989).

The recent studies by Huber et al also identified upstream sequences involved in the dexamethasone and IL- 6 stimulation of the jS-fibrinogen gene (Huber et al 1987). A

sequence determining dexamethasone response was far from the promoter, between - 2900 and -1503 from the transcription start point. The studies found a discrepancy between expression of the bacterial reporter gene chloramphenicol acetyl transferase (CAT) when driven by part of the j8-fibrinogen gene in response to dexamethasone and

/3-fibrinogen mRNA levels from HepG2 cells under dexamethasone stimulation. This suggests that there are either additional dexamethasone responsive elements in the jS-

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fibrinogen gene or increased messenger RNA stability under the action of dexamethasone (Huber et al 1990). The IL- 6 responsive elements were found to be

between -150 and -82 in the 5 '-flanking sequence of the gene and, since there were no differences between jS-fibrinogen mRNA analysis and CAT assays, this region was taken to contain all the IL- 6 responsive elements, although this may be a reflection of

the limited sensitivity of the in vitro assay used. As described above, the HNF-1 binding-site is in the same region so there may be some relationship between these two regulatory elements (Huber et al 1990). In the rat cK2-macroglobulin gene a short

sequence -CTGGGA- has been shown to be important in the IL- 6 mediated induction

of this gene (Ito et al 1989, M Hattori et al 1990, Hocke et al 1992) and this has been suggested to be a possible binding site for an acute phase transcription factor. This sequence is found in a number of acute phase reactant genes (Majello et al 1990, Oliviero and Cortese 1989, Poli and Cortese 1989, Wilson et al 1990, Won and Baumann 1990) and is present in all three fibrinogen genes and in the case of the jS- fibrinogen gene, this sequence is present within the sequence found to be required for IL- 6 stimulation in the studies of Huber et al described above.