Tabla de resultados

5.1 Introduction

The order o f the desmosomal cadherin genes on human chromosome 18 show that the position o f the genes correlate to the spatial order in which the DSC and DSG messages are expressed during morphogenesis of stratified epithelia (King et al.,

1997). These studies have been performed primarily on mice and they are consistent with the expression patterns seen using human foreskin sections. It has been proposed that an overall regulatory mechanism co-ordinates the regulation o f this gene cluster based on the close proximity of the genes and the direction o f transcription which extends away from an interlocus region (Cowley et al., 1997), possibly in the form of a locus control region. However, this hypothesis is based on the assumption that the order of the genes in human is the same as the order o f the genes in mouse which has not yet been established. Even though one would expect the order and orientation of the gene clusters to be the same in both species, the order in mouse needs to be determined.

The Whitehead Institute/MIT Mouse YAC Library (WI/MIT) was constructed at the Whitehead/MIT Center for Genome research, Massachusetts, USA using genomic DNA from C57BL/6J female mice (Haldi et al., 1996). The library was constructed by a ‘recombinational cloning’ method involving co-transformation o f target DNA along with two YAC vector arms carried on different plasmids, pRMLl and pRML2. The library represented a 10-fold coverage of the mouse genome with an average insert size o f 820 kb, and was available as DNA pools for PCR screening, which was a two step process to identify a positive clone. In the initial step 50 primary pools labelled MY1-MY50, each consisting o f eight pooled plates were acquired from the UK HGMP Resource Centre as DNA embedded in agarose. Following the instructions

provided, the agarose plugs were melted and screened by PCR. On the basis o f the primary pools, secondary pools were obtained and screened in the same manner.

5.2 RESULTS

5.2.1 Screening the WI/MIT YAC Library

Primers designed to the 5’ and 3’ ends o f the mouse desmosomal cadherins were used on genomic DNA to assess whether a specific band could be obtained by PCR for any of the 5’ or 3’ ends. None o f the primers gave a specific band. The cycling condition were varied but attempts were unsuccessful. It was then decided that the use of longer primers may increase specificity o f the PCR reaction. Primers originally used to screen a mouse genomic phage library (JC26 and JC28; I.E. Collins, unpublished data) were obtained and proved to be successful. JC26 and JC28 amplified a 5’ upstream region o f mouse DSC2 and were used to screen the 50 primary pools o f the mouse YAC library. 9 pools resulted in a band of the correct size (data not shown) but other weaker bands were also present. The pools were requested for further analysis (pools 7 ,9 ,1 1 , 22, 32, 33, 34, 45 and 48).

The secondary step consisted of:

• plated pools

• pooled columns corresponding to the columns from all plates o f the primary pools • pooled rows corresponding to the rows from all plates o f the primary pools

5.2.2 Identification of a YAC clone containing Mouse DSC2

The pooled plates, rows and columns of the secondary step were screened again using JC26 and JC28 primers. Only one o f the primary pools gave a positive result (Figure 5.1 A and B). Screening Pool 45 resulted in the identification o f plate 455 and coordinate 1 from the pooled columns. The coordinate for the row was vague and so YAC clones from co-ordinates B l, E l, G1 and HI o f plate 455 were requested. PCR

was performed on the four clones (Figure 5.1C) and YAC clone 455H1 was identified as a positive clone using JC26 and JC28 primers.

Further analysis will be required on this clone in order to determine the order o f the genes. In addition, it is not known whether this YAC clone has deletions or is chimaeric. PCR analysis using primers to DSCl or DSC3 was inconclusive (data not shown). The average size of the YAC clones obtained from this library is 820 kb. Thus it is conceivable that both the desmocollin and desmoglein clusters may be present on this YAC clone. The size o f the clone will also need to be determined.

Figure 5.1 PCR on YAC Primary pool 45

A Plate pools and pooled rows of the secondary screen Marker indicates 1 kb marker ladder

Lanes 451 -458 secondary plate pools

Blank no sample

Lanes 45A-45H pooled rows from primary pools

B Pooled columns of the secondary screen Lane M indicates 1 kb marker ladder

Lanes 45-1 to 45-12 pooled columns from the primary pool + PCR on genomic DNA using JC26 and JC28 primers

C YAC clones requested for further analysis Lane M indicates 1 kb marker ladder

Lane 1 YAC

Lane 2 YAC

Lane 3 YAC

Lane 4 YAC

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CD