The Weizmann Institute of Science Crown Human Genome Center

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Recent Large Scale DNA Sequencing Projects

There are three major projects currently ongoing at the LSDS unit:

 
DNA SEQUENCING IN THE 350 kb OLFACTORY RECEPTOR GENE CLUSTER ON HUMAN CHROMOSOME 17.
E. Ben-Asher, G. Glusman and D. Lancet.
Department of Molecular Genetics and the Crown Human Genome Center, The Weizmann Institute of Science, Rehovot 76100, Israel.

 The human olfactory subgenome represents several hundred olfactory receptor (OR) genes in a dozen or more clusters on several chromosomes. One OR gene cluster on human chromosome 17 (17p13) has been characterized in detail: it consists of at least 15 OR genes and pseudo-genes within a contiguous  stretch of approximately 350 kb of DNA. Using an OR-specific probe, 70 OR-positive cosmids were isolated forming a contig covering this region (Ben-Arie et al., 1994). In order to sequence this whole region, about 10 contiguous cosmids should be analyzed. The complete DNA sequencing of three cosmids within this cluster (cos39, cos73, cos17) has been accomplished by us. In addition to the confirmation of the expected OR genes, two new OR pseudogenes were identified. The location of all the repetitive sequences including  Alu, MER, L1 etc. have been determined. By similarity search we have identified, within this cluster, two large duplicated segments, that include OR genes, one of 11kb and another of ~30kb . These duplication events enabled us to suggest a model of  a general mechanism for genomic reorganization, explaining the process by which the OR repertoire may have expanded in mammals. 
 Moreover, similarity search of sequences flanking the coding regions of similar OR genes has enabled us to identify some regulatory signals of the OR genes' expression. (Glusman et al., 1996). For further confirmation of this model additional OR genes flanking regions will be sequenced and analyzed. 
 
 
 
 
CHARACTERIZATION, SEQUENCING AND FUNCTION ANALYSIS OF THE UPSTREAM
REGULATORY REGIONS OF THE HUMAN AML1 AND AML2 GENES.
 Ditsa Levanon, Thorsten Bangsow, Carmen Anghel, Amir Posner, Nir Rubins,
Edna Ben-Asher, Doron Lancet and Yoram Groner. 
Dept. of Molecular Genetics and the Genome Center. The Weizmann Institute of Science, Rehovot 76100, Israel.

 The human AML1 and AML2 genes belong to a recently identified gene family of transcription factors sharing a highly conserved region of 128 aa termed the "runt domain". The family includes the Drosophila segmentation gene runt, the human genes AML1, AML2 and AML3 and their respective mouse homologues. The AML1 gene resides on chromosome 21 and is involved in several leukemia associated translocations. AML2 resides on chromosome 1p36. Both AML1 and AML2 are involved in hematopoiesis, bind DNA and their binding is enhanced by heterodimerization with the Core Binding Factor beta protein (CBFb). 
 
 Large variety of isoforms of AML1 and AML2, resulting from differential promoter usage and from alternative splicing, have been characterized. These isoforms differ from each other by their 5' and 3' untranslated regions and by the length of their coding sequences. AML1 and AML2 bind the same response element and may therefor activate transcription of the same genes. Several forms of acute leukemia occur in children with Down syndrome (DS) at a 10 to 18 times higher incidence. The extra copy of chromosome 21 found in DS patients, is believed to be the causative agent of leukemia. The analysis of mRNA isolated from DS babies' blood revealed that AML2 was down regulated. Furthermore AML2 was significantly down-regulated in patients with chronic myeloid leukemia (CML) acute  myeloid leukemia (AML) and with acute lymphoid leukemia (ALL). Taken together, these data suggest that variations in the level of AML2, as a result of alteration in AML1 expression, could contribute to the transformation of cell of the hematopoietic lineage. 

 In order to better understand the involvement of AML2 in leukemogenesis, our lab investigate how variations of the level of AML1 expression in different form of leukemia can affect the expression of AML2 at a transcriptional level. We characterize and isolate the promoter regions of  the AML1 and AML2 genes and examine, both in vivo and in vitro, how the transcriptional regulation of these genes can be affected by changes in the physiological condition of the cell. AML1 and AML2 cDNAs with different 5' UTR have been characterized and used to identify the existence of two different promoters in each of these genes. Genomic mapping shows that these two promoters are located at a considerable distance from each other. 

 Two principal objectives are currently pursued: (1) structural and (2) functional investigation of uncharacterized AML1 and AML2 genes regions between and beyond the defined promoters. Using a linked reporter-gene system, the functional components of AML1 and AML2 regulatory regions will be further defined and analyzed. This will be done both in vitro and in vivo using mice transgenic for various AML1 regions defined by large-scale DNA sequencing. 

 With current levels of M13-shotgun sequencing technology at the Weizmann Institute, augmented by robotics devices and a well-established in-house automated fluorescent DNA sequencing facility, we envisage cosmid-to-readable-sequence data time-frames of 1-3 months per cosmid. Our intention is to proceed simultaneously from the proximal and distal promoter regions of the two genes in the telomeric direction. Sequence data will then link the proximal and distal promoter regions, and identify any 
regulatory regions telomeric to the distal promoter which may additionally be involved in AML1  and AML2 transcription. 
 
 

THE GENOMIC ENVIRONS OF THE DROSOPHILA SERINE/THREONINE KINASE GENE ia1.
Benny Motro, Aderet Reich, Shlomit Mesilaty, Ami Yannai, 
Aviva Moses, and Ron Wides. 
Department of Life Sciences, Bar-Ilan University, Ramat Gan, Israel.

        aurora (in Drosophila) and IplI (in yeast) represent two founding members of a growing serine-threonine kinase sub-family involved in controlling centrosome and chromosome segregation. IplI-aurora-like-kinase (ial) is a novel gene which represents a new 
member of this sub-family.  In order to establish the outer boundaries of this gene before constructing a complete transgene rescue element, the genomic environs of the ial gene were sequenced.   A 16 kb EcoRI genomic fragment was fully sequenced, followed by mapping of corresponding transcripts. 
 
        The ial transcript is closely bounded by transcripts of two other genes.  The transcription start site of a homolog of the mammalian STAM gene lies 500 bp from the transcription start site of ial.  The STAM gene encodes an adaptor protein which potentiates JAK signalling.  The two Drosophila genes, which are transcribed from opposing strands, appear to share transcriptional control elements, based on their proximity, and based on evidence of co-ordinate expression.  At the 3' end of the ial gene, a homolog of the C. elegans reading frame zk757 begins transcription less than 200 bp from the end of the ial transcript.  This transcipt has the capacity to encode a protein with motifs reminiscent of tyrosine phosphatases.  Immediately downstream of the zk757 homolog lies the gene 
for mitochondrial porin protein.  The remainder of the genomic sequence represents several kilobases which may represent a fifth gene. 

        The large scale sequencing of this genomic region, embarked on to clarify the boundaries of a single gene, has uncovered a number of genes with potentially relevant functions in the framework of our interests.  We are simultaneously using more than one of these genes as transgenic rescue elements.  This will allow us to carry out genetic screening in parallel with flies carrying these transgenes in order to identify mutations in this region which represent lesions in these genes.  Thus we will have direct proof of the roles of these genes. 
 
 

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Tsviya Olender, lvzvia@bioinformatics.weizmann.ac.il.