Evaluación de la ultrasonografía en la determinación de endometritis 46

4.1. INTRODUCTION.

D esp ite the g ath erin g e v id e n ce th a t m essag e sta b ility has a m ajor role to play in regulating m RNA levels, regulation at the level o f transcription is the norm al m ethod o f control o f m any cellular genes. The level of mRNA can be altered at two stages in the tran scrip tio n a l p ro cess, at the b asal lev el and at the reg u la te d lev el. T he b asal lev el is g o v ern ed by p ro m o te r seq u en ces p ro x im al to the tra n sc rip tio n in itia tio n site (T IS ), w h ilst d istal sequences called enhancers c o n tro l th e reg u lated transcription levels (see K adonaga, 1990 for a review ).

The prom oter elem ents are resp o n sib le fo r the assem bly o f the tran scrip tio n com plex. The consensus sequence T A T A A A or the T A T A box is the m o st com m on tra n sc rip tio n in itia tio n pro m o ter sequence in eukaryotes. T he tran scrip tio n com plex is assem bled at the TA TA site w hich is usually located 25 - 32 nucleotides from the transcription in itiatio n site. T he assem bly o f the com plex takes place in a stepw ise m anner starting with the TA TA box facto r TFIID and in v o lv e in co rp o ratin g several other transcription factors and the RNA polym erase II.

4.2 Transcription from TATA-less promoters.

N ot all prom oters contain TA TA boxes. T hose genes lacking T A T A boxes or T A T A -less gen es can be g ro u p ed in to tw o categories depending on the GC content o f their prom oters. The GC ric h p ro m o te rs g e n e ra lly h a v e m u ltip le tr a n s c r ip tio n in itiatio n sites and several S P l sites or G C boxes (Ish ii et al., 1 9 8 5 ), w h ile n o n -G C ric h p ro m o te rs h a v e o n e o r few tran scrip tio n in itiatio n sites. T he rat tPA gene is classed as a T A TA -less gene due to the absence o f a canonical TA TA box in its prom oter (Feng et al., 1990). It can be fu rth er categorised as a non-G C rich T A T A -less gene due to the low GC co n ten t (53% ) o f its prom oter and transcription in itiatio n from a single site. The m ouse tPA gene is also a T A T A -less gene, but the hum an tPA gene contain a consensus TATA elem ent (TA TA A A ). The rat pro m o ter has 78% and 55% hom ology to m ouse and hum an p ro m o te rs resp e c tiv e ly .

In the absence of a TATA box transcription initiation o f the rat tPA gen e has been found to tak e p lac e th ro u g h the S P l elem en ts in its p ro m o ter (fig u re 4.1). R esu lts from in vitro

stu d ies su g g est th at S P l elem en ts m ay act as tra n sc rip tio n in itiatio n elem ents in the rat tPA pro m o ter. O h lsso n et al. (1993) dem onstrated that the tw o S P l sites proxim al to the TIS are req u ired for the co n stitu tiv e ex p ressio n o f tPA in the rat neuroblastom a cell line B103 and fo r FSH m ediated induction of the gene in rat granulosa cells. They also found inactivation of the GC boxes alm o st ab o lish es the tPA p ro m o ter activ ity in th ese cell lines. F u rth erm o re, in the m u rin e tPA p ro m o te r

w hich is also a T A TA -less gene, the S P l sites w ere also shown to be essential both liver and brain specific expression o f tPA (P ecorino et al., 1991). T herefore the ev id en ce p o in ts to the S P l sites a d ja c e n t to the T IS a ctin g as th e tra n s c rip tio n initiation sequence in the rat gene.

A nother elem ent proposed to in itiate tran scrip tio n from non-G C ric h T A T A -le ss p ro m o te rs is th e in itia to r e le m e n t (In r), o rig in ally descried by Sm ale & B altim ore (1989). T he 17 bp In r o f the m ouse T dT gene w hich trav e rses its tran scrip tio n in itia tio n site w as fo u n d to re g u la te th e b a sa l le v e l o f tran scrip tio n of the gene. R ecom binant clo n e studies show ed that the Inr could be strongly activated by a TA TA box or by a h e te ro lo g o u s p ro m o te r in th e a b se n c e o f a T A T A box. F u rth erm o re, upstream p ro m o ter elem en ts w ere also found to d ire c tly a c tiv a te In r m ed iated tra n s c rip tio n w h ile m u ta tio n s w ithin the Inr elem ent affected the efficien cy o f its in itiatio n . T h erefo re these authors proposed th at the In r as the m inim al sequence elem ent that can initiate transcription from a gene.

T he p ro m o te r sites o f the a d e n o v iru s m a jo r-la te p ro m o te r (A dM L), rab b it 6-globin and hum an im m u n o d e fic ie n c y virus 1 w hich in itiate tran scrip tio n from in activ e T A T A -like sequences have been found to have hom ology to the m ouse T dT Inr. H ow ever, there is no hom ology betw een the Inr and the rat or m ouse tPA start sites, but it is possible there m ay be d ifferen t types o f In r elem ents in differen t prom oters w hich can in itiate

transcription in association with upstream sequence elements.

4.3 T he ro le o f enhancers in tran scrip tio n regulation.

T he re st o f the pro m o ters o f ra t and m ouse tPA genes also d iffer in th eir p o ten tial b inding sites fo r tran scrip tio n facto rs. The rat gene has one A P I, one C T F /N F l, one CRE and three S P l sites (inclusive o f the initiation elem ent) in its p rom oter (figure 4.1), w hile the m ouse tPA prom oter consists o f five A P2s, one S P l, one TRE and one C T F/N Fl site.

T ran scrip tio n factors (TFs) that bind to these elem ents in teract w ith the tran scrip tio n com plex assem bled at the TIS to alter the level o f transcription of the m essage. O f the several m odels p u t fo rw ard to e x p la in the in te ra c tio n s b etw een the fa c to rs bound to the d istal enhancer elem ents and those bound to the p ro x im a l p ro m o te r elem en ts the lo o p in g m o d el is the m o st w idely accepted theory now. This m odel suggest th at once the TFs have bound to the enhancer elem ents the DN A folds (loops- out) to bring these into close co n tact w ith the basal regulatory fa c to rs. S e v e ra l v a ria tio n s to th is m o d el h as also been proposed (Ptashne & Gann, 1990) (figure 4.2).

The regulatory factors can be grouped into universal factors e.g. S P l, w hich is expressed ubiquitously by m any cells and tissue specific factors, e.g. Oct 1 which is expressed in a tissue specific m anner. A lthough S P l can be classified as a universal factor as b inding sites fo r the fac to r is p resen t in m any p ro m o ters, it m ay n o t be alw ays involved in in itiatio n o f gene tran scrip tio n

as found w ith tPA.

T h e a f f in ity o f so m e p r o m o te r e le m e n ts to m u ltip le tran scrip tio n facto rs leads to th e ir genes b ein g reg u la te d in d isp arate m anner in d ifferen t tissu e. F o r ex am p le, the PEA 3 en h an c er e lem en t in the uPA p ro m o te r h as been show n to in flu e n c e u P A g en e tra n s c rip tio n in a d if f e r e n t m a n n e r depending on the cell line. N erlov e t al. (1991) studied PEA3 m ed iate d uPA e x p re ssio n in th ree hum an tra n sfo rm e d c e ll lines; PEA3 was found to induce highest levels o f uPA in HepG 2 hepatom a cells, it was less active in the H T 1080 fibrosarcom a cell line and was inactive in the third, H eLa cell line.

The m urine tPA gene can also be expressed in a tissue specific m anner (P eco rin o et al., 1991). T hese w o rk ers used an i n v i t r o assay to in v estig ate the effects o f m urine brain, kid n ey and liv er extracts on the expression o f tPA m RN A. T he brain had the h ig h est steady state lev els o f tPA m R N A w hile the kidney had low er levels and it w as u n d etectab le in the liv er.

In v itro tra n s c rip tio n assay s u sin g e x tra c ts fro m the th re e sites show ed h ig h est lev e ls o f tra n sc rip tio n fro m the b rain . T h ere w as lo w er lev els o f tra n sc rip tio n in th e p resen c e o f kidney ex tracts and even low er but d e tectab le tran scrip tio n in the liv e r. T hese d ata su g g est th at tra n sc rip tio n rate s c o u ld partly account for the differences in the tPA levels betw een the th ree sites. F u rth e rm o re , m o b ility sh ift a ssa y s d u rin g th is study found sp ecific b rain d eriv ed facto rs th at had in creased affin ity to the GC boxes or to sequences o v erlap p in g the GC

5P CRE