2.7. MATERIALES USADOS EN ESTRUCTURAS METÁLICAS
2.8.2. BASE DE DISEÑO
The central dogm a of m olecular biology is 'D N A -R N A -Protein'. This dogm a states th at DNA is transcribed into RNA and RNA is then translated into protein. The transcription of DNA into RNA is m ediated by the enzym e RNA polymerase. In eukaryotic organism s there are three classes of RNA polym erases, nam ely RNA polym erase I, II and III. T ranscription of mRNA from genes w hich encode proteins are m ediated by RNA polym erase II. The regulation of transcription of mRNA from a p a rticu la r gene requires the coordinated action of specific p ro tein s or transcription factors (discussed below).
1.16.1 Transcription
The in itiatio n of RNA polym erase II m ed iated tran scrip tio n of eukaryotic genes requires the interaction of transcription factors th at bind
DNA. These tran scrip tio n factors bin d D N A in a sequence-specific
m anner. The specific DNA sequences (elem ents or m otifs) to w hich
proteins bind can be in close proxim ity or extremely distant to a particular gene. In m ost cases the DNA elem ents involved in these interactions are
clustered together w ithin the 5' flanking region of the gene. The region of
the DNA w here these transcription factor binding sites are found is term ed the prom oter. M any different transcription factors have been identified, w h ic h are in v o lv ed in th e p ro cess of c o n stitu tiv e or e n h a n c e d
tran scrip tio n . The p ro p erties and b in d in g sequences of these p ro tein
and M cKnight, 1989). The following sections describe the function and properties of a few of the know n transcription factors.
The TATA box, an AT-rich sequence (consensus T A T A A /T A A /T ), is found about 30 base pairs upstream of the transcription start site of m ost eukaryotic genes. The TATA box is necessary for the correct positioning of RNA polym erase at the start site for the initiation of b asal levels of
tran scrip tio n (B uratow ski et ah, 1988). The rate of transcription can be
enhanced considerably from its initial basal level by the b in d in g of other transcription factors to specific sites upstream of the TATA box. These factors m ay be tissue-specific or be induced by a particular stim ulus or act in a constitutive m anner. C onstitutive factor binding sites are found in a diverse n u m b er of genes. Two of the w ell characterised constitutive transcription factor binding sites are the CCAAT and GC (or S pl) boxes (Santoro et ah, 1988; R upp et ah, 1990; Dynan and Tjian, 1983; G idoni et ah,
1984). The transcription factors CTF (CCAAT box transcription factor) and Spl bind to the CCAAT and GC boxes, respectively.
The S p l p ro tein binds to the consensus sequence GGGCGG. The S p l protein is found in all cell types. Spl protein contains tw o glutam ine- rich dom ains w hich interact w ith the RNA polym erase II and associated proteins (Latchman, 1995). These glutam ine rich dom ains are essential for the activation of transcription of genes which contain the Spl-binding site. The D N A -binding ability of the Spl protein is d ep en d en t on three zinc finger m otifs, situated w ithin the C -term inal region of this transcription factor (Berg, 1992). Zinc finger m otifs are found in other transcription factors. The zinc finger is a 30 amino acid repeat unit of w hich 12 residues form a loop, w hich projects out from the surface of the p ro tein (Figure 1.12). The amino acid loop is anchored at its base by tw o cysteine and two histidine residues, which bind a zinc atom (Figure 1.12). These tw o Cys an d H is residues are conserved in all zinc finger m otifs. Zinc fingers facilitate protein to DNA binding. The S pl protein requires the presence of zinc for its sequence-specific binding activity.
There are several different proteins w hich bind to the CCAAT box
(R ajm o n d jean et ah, 1988). Some of the p ro tein s w hich b in d to the
CCAAT box can be expressed in a tissue-specific m anner, w hereas others are expressed in all tissues (reviewed Johnson and McKnight, 1989). One of the first CCAAT box b in d in g p ro tein s to be id en tifie d w as the transcription factor CTF (Rosenfeld and Kelley, 1986). In contrast to the glutam ine-rich activation regions of S p l, the factor CTF contains proline- rich activation dom ains (Latchm an, 1995). The proline-rich dom ain of
His Cys Zn Cys His His Cys Zn Cys His Protein surface
Figure 1.12 Schematic representation of two zinc finger motifs
(adapted from Latchman, 1995). The finger motifs project out
from the surface of the protein. A zinc atom is bound at the base
of each motif by conserved cysteine and histidine residues.
CTF is situ ated w ith in the C -term inal region of its protein. P roline resid u es constitute about 25% of the am ino acids found w ith in the C- term inal region of the CTF protein. Proline-rich dom ains are fo u n d in
o ther tran scrip tio n factors, such as AP-2, c-Jun and Oct-2 (M itchell and
Tjian, 1989). This proline-rich dom ain is required for the activation of
transcription (Gerber et a l, 1994).
Some genes m ay have both of these S p l and CCAAT box DN A elem ents, w hereas others have single or m ultiple copies of one or the other, w orking in a concerted fashion to regulate the rate of transcription. T hus, th ese co n stitu tiv e factors play an essen tial role for efficient transcription of the gene to occur.
1.16.2 Enhancer elements
O ther DNA sequences located at a distance from a gene (100 bp to several 1000 bp) w hich influence the activation of transcription are term ed enhancer elem ents. Enhancer elem ents bind transcription factors w hich in tu rn influence the rate of tran scrip tio n from a p a rtic u la r gene. E nhancer elem ents are usually found w ithin the 5' flanking region of a gene. W here enhancer elem ents are in close proxim ity to the TATA box, w ith in the 5' flanking DNA, this region is term ed the proxim al prom oter. V arious w orkers studying enhancer elem ents have established th at these DN A sequences possess three basic properties. These p roperties are 1; enhancer elem ents can influence a prom oter regardless of the orientation of its seq u en ce to th e gene, 2; e n h an c er elem en ts can in flu en ce tra n sc rip tio n equally w h eth er it is w ith in the proxim al p ro m o te r or several 1000 bp aw ay from the prom oter, 3; the enhancer elem ent can affect activation of transcription w hether situated w ithin the 5' or 3' end of the transcribed region (Latchman, 1995). In some cases enhancer elem ents
can also be found w ithin the intron of the gene it influences (Singh et al.,
1986). M any enhancer elements act in a tissue-specific m anner. That is the enhancer elem ent of a particular gene which is expressed only in a specific tissue-type will only have a stim ulatory effect on genes w ithin th at tissue and not on any other tissue (see Section 1.16.3).
1.16.3 Tissue-specific expression
In eu k ary o tes the p rin cip al m eans of controlling tissue-specific production of proteins is by the regulation of the initiation of transcription (Latchm an D, 1995). Regulation of transcriptional initiation is achieved by the production of certain protein factors w hich are exclusive to a particular
tissue or even to a specific cell-type. H ow protein factors can confer tissue- specificity is described below.
One of the best examples of tissue-specific gene expression is for the
im m u n o g lo b u lin genes. There are tw o im m u n o g lo b u lin genes, one
encodes for a heavy chain protein and the other for a light chain. Both of these genes are expressed exclusively in B lym phocyte cells. Both the h eav y - an d lig h t-ch ain genes attain th eir B cell specificity via the interaction of B cell-specific factors, w hich bind to the prom oter region. Both of the im m unoglobulin genes contain a conserved octam er sequence 5'-ATTTGCAT-3' w ithin their 5' flanking prom oter regions, w hich binds
the protein factor Oct-2 (Singh et ah, 1986). Sequence specific b in d in g of
Oct-2 confers B cell-specific enhancem ent of expression of the light- and
heavy-chain genes (Wirth et al., 1987). The tissue-specificity of expression
arises from the fact that Oct-2 is transcribed and translated, at high levels, in B lym phocytes but not in m ost other cell types.
It is im portant to note that the octamer sequence in the heavy-chain gene p ro m o ter is in an inverted orientation (5'-ATGCAAAT-3') to the octam er sequence in the prom oter of the light chain gene (Figure 1.13). This exam ple perfectly illustrates one of the properties of a DN A motif. That is, the function of the DNA motif to act as a protein binding site can be in d ep en d en t of its orientation. M utation or rem oval of this octam er m otif from the prom oter region of the light or heavy genes elim inates B-
cell-specific expression (Gerster et ah, 1987). Indeed, placing this octam er
sequence w ith in a non-im m unoglobulin gene prom oter results in B cell-
specific expression of this altered gene (Wirth gf al., 1987). Thus, the Oct-2
sequence acts as a tissue-specific enhancer elem ent to confer B lym phocyte- specific expression.
1.16.4 Photoreceptor-specific expression
V isual pigm ents are found exclusively in photoreceptor cells (see Section 1.7) and expression studies of photoreceptor genes have show n
th at they are regulated in a tissu e/ cell-specific m anner (Lem et al., 1991).
Thus, it w o u ld be logical to assum e th at the prom oter regions of visual p ig m en t genes, and other photoreceptor-specific genes, contain DN A motifs that confer this specificity.
The use of reporter constructs in transgenic mice has allow ed the
determ ination of the m inim um 5' flanking region of a gene required for
cell-specific expression in the retina. A 1.3 kb fragm ent from the 5' end of
Initiation ATG Heavy 5'ATGCAMT3' Light 5'ATTTGCAT 3 ' V Exon 1 Intron 1 ►
Octamer TATA 5'UTR
Figure 1.13 The immunoglobulin heavy- and light-chain gene proximal promoter region (adapted from Latchman, 1995). The promoter contains a TATA box and an octamer motif. The mRNA initiation start site and the ATG start codon is indicated. The 5' untranslated region (5'UTR) and exon one of the gene is also shown. Note that the octamer is in an opposite orientation in both genes.
can direct lacZ expression specifically to rod and cone cells (Yokoyama et
al., 1992). Transgenic mice containing a shorter hum an interphotoreceptor
retinoid-binding protein gene prom oter fragm ent of 141 bp has recently been show n to confer tissue specific expression to retinal photoreceptors
an d pinealocytes (Bobola et ah, 1995). Transgenic mice w ith bovine rod
opsin prom oter constructs have show n that fragm ents as sm all as 292 bp or as large as 2244 bp (both constructs include 70 bp of 5' u n tran slated region) are adequate enough to direct photoreceptor specific expression (Zack et ah, 1991).
W hich sequences direct cone-cell-specific expression as opposed to rod-cell-specific expression? A 37 bp region term ed the locus control region (LCR) w ith in the h u m an RCP gene pro m o ter w as show n to be
essential for cone-specific expression in transgenic mice (Wang et ah, 1992).
p -g alacto sid ase rep o rte r constructs con tain in g the h u m a n RCP gene
p ro m o te r w ith its LCR d eleted , show ed no ex p ressio n of the l a c Z
transgene. H ow ever, the transgene of the hum an RCP gene prom oter plus its LCR gave expression to both types of cones, the blue and the re d /g re e n
cells, w ith in the m ouse retin a (W ang et ah, 1992). This resu lt w as
unexpected given the lack of sequence hom ology betw een the hum an BCP and R C P /G C P gene prom oter. It is possible th at the h u m an RCP gene prom oter construct, used in the above study, m ay lack certain regulatory elem ents w hich repress the expression of the R C P/G C P gene in m ouse
blue cones (Wang et ah, 1992).
A 6.4 kb DNA fragm ent derived from m ouse BCP prom oter, linked
to a p-galactosidase {lacZ ) reporter construct, was able to target expression
exclusively to blue cones in transgenic mice (Chiu and N athans, 1994a). A sim ilar experim ent using 3.8 or 1.1 kb of the hum an BCP gene 5' flanking region gave reporter expression in blue cones, as w ell as in som e cone
b ip o lar cells, in transgenic mice (Chen et ah, 1994). A sm aller reporter
construct containing only 470 bp of the h u m an BCP gene prom oter w as also found to give expression to bo th blue cones and a subset of cone b ip o lar cells (Chiu and N athans, 1994b). This difference in expression patterns betw een the hum an and m ouse BCP prom oter constructs could be due to a variation of regulatory sequences betw een the m ouse & hu m an transgene fragm ents (Chiu and N athans, 1994b). The above experim ents im ply th at only 470 bp of 5' flanking region from the h u m an BCP gene is required for its blue cone cell-specific expression. Thus, the DNA sequence m otifs responsible for this cell-specific expression sh o u ld be contained w ithin this short prom oter region.
The LCR and the recent discovery of other photoreceptor-specific c/s-acting elements are described in Section 5.1.2.
1.17 Aims of the project
• W hich amino acid residues w ithin the S group of pigm ents are
responsible for their particular Xmax?
To isolate and characterise a non-prim ate m am m alian blue cone pigm ent (BCP) gene belonging to the S group of visual pigm ents, nam ely the porcine BCP gene.
To com pare the predicted porcine BCP protein w ith other m em bers of the S group, to attain a better understanding of the visual pigm ents w ithin this group.
To predict potential spectral tuning sites, which m ay be responsible for causing shifts in Xmax/ from a com parison of the S group of pigm ents w ith other vertebrate pigm ents.
• To test the feasibility of using a yeast {Pichia pastoris) expression
system for the expression of hum an opsin genes.
• W hich czs-acting regulatory elements direct cone-cell-specific
expression as opposed to rod-cell-specific expression?
To isolate and sequence the proxim al 5' flanking region from prim ate and porcine BCP genes.
To characterise these isolated m am m alian BCP gene 5' flanking
regions for evolutionary conserved czs-acting motifs.
To characterise the WERI-Rbl cell line. To establish w hether or not this retinoblastom a cell line is expressing transcripts for the
h u m an BCP gene.
To isolate nuclear proteins from W eri-Rbl cells, cultured in vitro,
w hen BCP gene expression is confirmed.
To show that these W eri-Rbl nuclear proteins could be used for electrophoretic mobility band shift assays on fragm ents of the h u m an BCP gene prom oter.
• To localise the hum an BCP gene to a specific region of hum an
chrom osom e 7.
To characterise a genomic cosmid clone containing the h u m an BCP gene.