Determinación de la calidad bacteriológica de las aguas de pozo de los

S6 has sequence identity with a recently cloned mouse gene, neuronal leucine rich repeat protein 1 (NLRR-1) (Taguchi et al, 1996). This was isolated after an EST clone from a human brain cDNA library that contained leucine rich repeats (EST 06184) was used to screen a commercially available neonatal mouse brain cDNA library

(Stratagene). The EST was picked from the database on account of its homology to the drosophila gene slit, which plays a role in central nervous system development {vide infra). A full length 3.7 Kb clone (NLRR-1) was isolated by this screen as well as a 217bp EST named NLRR-2 which encoded an iteration of several related LRRs. Rudimentary analyses of the in situ hybridisation expression patterns of these species was performed in sections of 13.5 dpc embryos and adult brains. NLRR-1 had wide ranging expression throughout the embryonic nervous system and also in the

hippocampus and cerebellum of the adult. No evidence of somitic expression can be seen in the published in situ hybridisation images. NLRR-2 also showed expression in the adult brain but no clear embryonic expression could be seen.

The same group subsequently cloned by the same method a third related full length species named NLRR-3 (Taniguchi et ai, 1996). Like NLRR-1, this contained 11 LRRs with amino and carboxyl flanking cysteine rich repeats, a 5' signal peptide sequence and a 3' transmembrane domain. Similar rudimentary in situ hybridisation studies showed signal in the developing central nervous system (dorsal root ganglia, thalamus and nasal epithelium) of 13.5 dpc embryos. No somitic expression could be determined from the published data. In the adult brain expression was restricted to hippocampus, dentate gyrus and olfactory bulb.

Using wholemount in situ hybridisation and studying much earlier post - gastrulation embryos, the striking areas of NLRR-1 expression are in the somite and the midline of the forebrain (figure 4.3a). Later embryos than the 30 -35 somite stage (10.5 dpc) were not studied as the technique is generally not amenable to these stages. Recently, section data (not presented here) have shown the gene to be expressed in migrating cells of the dorsal myotome.

6.5.2.1 The isolation of novel NLRR homologues from the mouse embryo

A simple protein alignment of the sequences of NLRR-1, NLRR-2 and NLRR-3 obtained from the GenBank database allowed the generation of primers corresponding to conserved sequence motifs (KELG and GDFX) for use in 5' RACE PCR with RNA from somites 5 - 7 of 25 somite stage embryos. Initially this was performed with dATP tailing as no satisfactory upstream consensus could be found. A novel NLRR family member named LRR-4 was isolated and shown to have a classically myotomal pattern of expression (figure 4.9) from the 15 - 20 somite stage. Detailed sequence analysis of LRR-4 suggests that it is the full length clone of the gene represented by the short NLRR-2 EST.

A full alignment of the protein sequence of LRR-4 with those of NLRR-1 and NLRR-3 has allowed for the generation of a primer corresponding to conserved sequences in the amino flanking cysteine rich sequences of these genes. Using this in conjunction with the GDFX primer in PCR of whole embryo (20 - 25 somite stage) RNA which had been reverse transcribed with the KELG primer, two further members of the family have been isolated. One is NLRR-3 and the other has DNA sequence identity with an EST derived from a mouse placenta cDNA library (EST aa020425). In addition to this, the EST database has been searched unsuccessfully using the BLASTn algorithm for further homologues.

6.5.2.2 Homologues of the NLRR genes in drosophila

The two previously cloned full length NLRR genes and the novel gene LRR-4

presented here all contain iterations of an LRR motif most closely related to those of the drosophila genes slit and tartan (Rothberg e ta l, 1988; Chang et a l, 1993). Both of these genes encode LRR containing molecules important in different cell signaling processes in the embryology of the fly.

Slit, which also contains seven EGF repeats at its carboxyl terminus is a secreted protein produced by midline glial cells of the developing embryo. It has four separate tandem arrays of LRRs each of which has its own amino and carboxyl flanking cysteine rich motifs. It appears to play a role in the interactions between midhne glial cells, the extracellular matrix and decussating axons (Rothberg et a l, 1990).

Tartan, on the other hand is a transmembrane molecule with a similar overall structure to the NLRR family. It has a single array of 14 LRRs downstream of its signal

sequence which is flanked by single amino and carboxyl terminal flanking cysteine rich domains. At the carboxyl end, it has a transmembrane domain. Work on this gene in the fly is scant, but it appears to be broadly expressed in the nervous system and in developing muscles. The fly mutants of this gene have disrupted myogenesis amongst their many phenotypic traits (Chang et a l, 1993).

Despite their broad structural similarity to tartan, the NLRR genes have greater primary sequence homology to the LRRs and flanking domains of slit. It must be reiterated however that they are transmembrane molecules and do not have EGF repeats.

6.6 Conclusions and future directions

A modified differential display protocol has been developed which can be reliably used to detect transcript differences between single dissected murine embryonic structures using wholemount in situ hybridisation as a secondary screen. Using this method, a novel somitic marker which has been recently cloned from the adult mouse brain has been isolated. This is a leucine rich repeat transmembrane protein, NLRR-1 which whilst structurally analogous to ihodrosophila g^nctartan, has greater primary sequence homology over its LRR and flanking domains toslit. Using low stringency PCR and 5' RACE, a novel related gene, LRR-4 has been isolated which is expressed in a different domain of the somites.