Mecanismo de reacción para la formación del compuesto 3.

Another mammalian origin of DNA replication has been identified by studying the direction of DNA replication covering more than 200 kb across the human p-globin locus which encodes six p-like globin genes. A single bidirectional origin o f DNA replication has been located upstream of the p-globin gene using this method. This

site was narrowed down to a defined 2 kb DNA fragment (Kitsberg et a l , 1993).

This locus is used to direct DNA replication in both erythropoietic cells which actively transcribe the globin genes and in cells which do not (Kitsberg et a l , 1993).

In cells which do not actively transcribe the globin genes, replication o f the locus occurs late, while transcribing cells replicate the locus early (Epner et a l , 1988;

Hatton et a l , 1988).

Patients suffering from the haemoglobin Lepore syndrome carry an approximately 8 kb deletion within the p-globin locus that includes the identified 2 kb replication

origin (Mears et a l , 1978). In cells derived from these patients, the direction of DNA

replication is reversed upstream of the origin locus, indicating that replication initiated from a site outside the studied locus (Kitsberg et a l , 1993).

50 kb upstream of the human P-globin replication origin lies the p-globin "locus control region" (LCR), which controls transcription, chromatin structure and

C hapter 1

replication timing of the entire locus (Forrester et a l , 1990). A targeted insertion into

the LCR locus which inactivated (3-globin gene transcription and the chromatin

structure of the locus did not affect initiation from the p-globin origin (Aladjem et a l,

1995; Kim et a l , 1992). In patients suffering from Hispanic thalassemia, a 35 kb

region of DNA encompassing the LCR has been deleted (Driscoll et a l , 1989). This

mutation inactivates the transcription of P-globin genes and causes a major alteration in chromatin structure (Driscoll et a l , 1989; Forrester et a l , 1990). Despite being

about 50 kb away from the p-globin replication origin, this deletion completely abolished initiation from the origin. Instead, the locus apparently replicated from a downstream initiation site providing evidence for requirement of distantly separated

sequences for initiation of DNA replication (Aladjem et a l , 1995).

To study the ability of the p-globin replication origin to function in novel chromosomal locations, an 8 kb fragment carrying the origin was transferred to ectopic sites in the simian genome. DNA synthesis indeed initiated from p-globin replication origin sequences located within the monkey chromosome, suggesting that specific sequences within this DNA fragment can function as a classic replicator. In these contexts, initiation from the p-globin replication origin was independent of the LCR which may act to alleviate a repressive effect of chromatin structure. In an ectopic location, this function may be served by other cis-acting elements.

The study of the 8 kb p-globin replication origin deletion mutants in ectopic location showed that deletions of 1.5 kb 5' or 4 kb 3' did not prevent initiation. However, a construct carrying both deletions; or a deletion of a 2.6 kb "core" region failed to initiate. The shortest fragment that was able to support initiation was 4.2 kb long and

Structure o f mammalian replication origins comprising o f "core" sequences and auxiliary elements that affect replication efficiency.

Initiation in Drosophila chorion cluster localises to the ACE

The chorion genes of Drosophila provide an unique opportunity to study the

replication of specific chromosome regions in a higher eukaryote. Chorion genes encode structural proteins of the eggshell and are arranged in a tandem cluster of four on chromosome three and a cluster of six on the X chromosome (Spradling, 1981; Spradling and Mahowald, 1980). During oogenesis, ovarian follicle cells over­ replicate the chorion genes, during the same cell cycle, producing high levels of eggshell proteins. The cluster on chromosome X over-replicates approximately 15- fold, while the cluster on the third chromosome amplifies about 60-fold; equivalent to four and six rounds DNA replication, respectively. Gene amplification on the third chromosome occurs by initiating multiple rounds of DNA replication in the vicinity

o f the four tandemly arranged genes (Figure 1/6). Each newly initiated fork

progresses along the chromosome, duplicating both the gene region and up to 50 kb of flanking chromosomal DNA, producing a bell-shaped gradient of amplification centred at the site o f the gene cluster (Spradling, 1981).

To study the ability of DNA sequences from the third chromosome chorion locus to initiate the additional rounds of DNA replication, DNA segments derived from the cluster were inserted into novel chromosomal sites using P-element mediated transformation. Only a transposon containing a specific 3.8 kb segment derived from the cluster underwent amplification during oogenesis (Figure 1/6). However, the ability o f this transposon to support initiation was subject to position effects. Attempts to induce amplification with subfragments o f the 3.8 kb segment were unsuccessful, suggesting that most of this fragment is required for amplification (de

Chapter /

Cicco and Spradling, 1984). Deletion analysis of this 3.8 kb fragment identified a 510 bp region upstream of the s l8 gene, which is essential for its function. This

Drosophila chorion cluster on chromosome three

chorion gene cluster

sl8 sl5 sl9 sl6

- O H

I

E Z I O

ACE A E R -d ( p ) AER-c AER-b AER-a

3.8 kb fragment

► - 4

7.7 kb fragment 4.2 kb fragment

F i g u r e 1/6. Organization o f the D rosophila chorion gene cluster on ch rom osome three. Arrows labelled s i 8, s i 5, s l 9 and s i 6 represent the tandemly arranged chorion genes. ACE is the amplification control element and AER a-d represent the amplification enhancing regions. The AER-d region is also known as (3. The 3.8 kb fragment was initially identified by its ability to confer replication competence at a novel chromosomal location. The majority of replication bubbles occured in the 7.7 kb fragment with smaller number of bubbles found in the 4.2 kb fragtiient.

region became known as the "amplification control element" (ACE) (Figure 1/6; Orr- W eaver and Spradling, 1986). However additional sequences outside of the ACE were found to enhance amplification. These "amplification enhancing regions" (AER) could support a low level of amplification even in the absence o f ACE, suggesting functional redundancy within the chorion replicon (Delidakis and Kafatos,

1987; Swimmer er «/., 1989).

The neutral/neutral 2D gel electrophoresis analysis of the chorion cluster revealed that the majority of the initiation events occurred within a 7.7 kb region with small number of bubbles found in the adjacent 4.2 kb fragment (Figure 1/6). However, multiple start sites within the 7.7 kb region were observed (Brewer and Fangman, 1987; Heck and Spradling, 1990). To clarify which DNA sequences were important

for the initiation o f replication, the 3.8 kb fragment was studied by two-dimensional electrophoresis in a novel chromosomal location. In this setting, the majority of the initiation events localised to the ACE region and to the previously identified AER-d region which is also known as (3 (Heck and Spradling, 1990).

In summary, the emerging picture of a replication origin in higher eukaryotes involves interplay between DNA sequences, which appear to have a modular structure and a high degree of redundancy, and regulation by chromatin structure. In

the case of Xenopus eggs however, DNA replication initiaties from random sequences

and the mechanism responsible for this phenomena remains unclear.