Duplication of a genomic region containing the Cdc2L1-2 and MMP21-22 genes on human chromosome 1p36.3 and their linkage to D1Z2

Abstract

Cdc2L1 and Cdc2L2 span approximately 140 kb on human chromosome 1p36.3. The products of the Cdc2L genes encode almost identical protein kinases, the PITSLRE kinases, which have functions that may be relevant to the regulation of transcription/splicing and apoptotic signaling. These genes are deleted/translocated in neuroblastomas with MYCN gene amplification, a subset of malignant melanomas, and in a newly delineated deletion syndrome. Here we report that the p36.3 region of human chromosome 1 consists of two identical genomic regions, each of which contain a Cdc2L gene linked to a metalloprotease (MMP) gene in a tail-to-tail configuration. This duplicated genomic region is also linked tightly to D1Z2, a genetic marker containing a highly polymorphic VNTR (variable number tandem repeat) consisting of an unusual 40-bp reiterated sequence. Thus, these genes and the polymorphic marker D1Z2 are organized as follows: telomere-D1Z2-5'-MMP22-3'-3'-Cdc2L2-5'-5'-Cdc2L1 -3'- 3'-MMP21-5'-centromere. Remarkably, the introns and exons of Cdc2L1 and Cdc2L2, as well as their flanking regions, are essentially identical. A total of 15 amino acid differences, 12 nonconservative and 3 conservative, can be found in the 773-786 amino acids specified by the various products of the Cdc2L genes. Two separate promoter/5' untranslated (UT) regions, CpG1 and CpG2, are identical to a reported previously methylated genomic CpG sequence and are used to express >20 different Cdc2L transcripts from the two genes. The expression of CpG2 transcripts from Cdc2L1 and Cdc2L2 is tissue/cell-line specific. CpG1 transcripts are expressed ubiquitously from both genes, with perhaps some bias towards the expression of CpG1 Cdc2L1 mRNAs in certain hematopoietic cells.

Figure 1

Schematic representation of the duplicated Cdc2L1/2 and MMP21/22 genes linked to D1Z2 on 1p36.3. Shown is the organization of two duplicated genomic segments on human chromosome 1p36.3; each segment contains a Cdc2L and MMP gene (Cdc2L1 and MMP2 or Cdc2L2 and MMP22). Also shown is their transcriptional orientation relative to one another, the mosaic structure of the Cdc2L genes (each having 21 exons and 20 introns), their linkage to D1Z2, and their relationship to telomeric and centromeric regions of the short arm of chromosome 1. The various human genomic cosmid, P1 phage, and BAC clones used to construct this detailed map are indicated below the map.

Figure 2

Detailed schematic comparing the relationship between the structure of the PITSLRE protein kinase and Cdc2L1/Cdc2L2. The structure of the PITSLRE protein kinase and the Cdc2L exons that encode the protein is shown in detail at top. Amino acid differences between the proteins encoded by Cdc2L1 and Cdc2L2 are indicated below the protein schematic. Directly below this schematic are representations of the various PITSLRE mRNAs (denoted as splice variants) generated by alternative splicing from Cdc2L1, Cdc2L2, or both.

Figure 3

Fiber FISH analysis of the Cdc2L cosmids and the chromosome 1p36.3 marker D1Z2. Fiber FISH analysis using (A) hCos8A and D1Z2, (B) hCos2A and D1Z2, (C) hCos9A and D1Z2, and (D) hCos6–3 and D1Z2. Cosmids 2A, 8A, and 9A contain Cdc2L2; cosmid 6–3 contains Cdc2L1. The D1Z2 probe is red and the cosmid DNA probes are green. Significant overlap is indicated by yellow, as in C.

Figure 4

PCR–LR analysis of Cdc2L1 and Cdc2L2 mRNAs expressed by various cell lines. An analysis of the expression of Cdc2L1 and Cdc2L2 using a unique EcoRI restriction site found in exon 17 of Cdc2L2 (bottom) was performed by RT–PCR. The undigested (−) and EcoRI-digested (+) RT–PCR products were separated by agarose gel electrophoresis, transferred to a membrane, and hybridized with the PITSLRE cDNA. Whereas most cell lines express both Cdc2L1 and Cdc2L2 transcripts, Daudi and U937 cells appear to express primarily Cdc2L1 mRNA, which lacks the diagnostic EcoRI site. (Bottom) A schematic of Cdc2L structure relative to the location of the PCR primers used in this analysis. The expected sizes of the undigested (539 bp) and digested (359 and 180 bp) products is also indicated.

Figure 5

RT–PCR analysis of the expression of Cdc2L transcripts originating from the two different CpG promoter regions in various human tissues and cell lines. (A) RT–PCR analysis of CpG1 and CpG2 (see Fig. 1) transcripts expressed by Cdc2L1 and Cdc2L2. The various tissues and cell lines are indicated above each lane, whereas five different sized products (see text for explanation) are indicated on the right of each panel. (B) RT–PCR analysis of the expression of CpG1 transcripts corresponding to the Cdc2L1 gene, using the same set of tissue and cell line RNAs. The arrows at right indicate that these CpG1 mRNAs are expressed in a few, but not all, of the sources examined. Decreased resolution of the two RT–PCR products (bottom) is caused by the short run of the agarose gel used to analyze the RNAs from the various cell lines.

Figure 5

RT–PCR analysis of the expression of Cdc2L transcripts originating from the two different CpG promoter regions in various human tissues and cell lines. (A) RT–PCR analysis of CpG1 and CpG2 (see Fig. 1) transcripts expressed by Cdc2L1 and Cdc2L2. The various tissues and cell lines are indicated above each lane, whereas five different sized products (see text for explanation) are indicated on the right of each panel. (B) RT–PCR analysis of the expression of CpG1 transcripts corresponding to the Cdc2L1 gene, using the same set of tissue and cell line RNAs. The arrows at right indicate that these CpG1 mRNAs are expressed in a few, but not all, of the sources examined. Decreased resolution of the two RT–PCR products (bottom) is caused by the short run of the agarose gel used to analyze the RNAs from the various cell lines.