발표자 석사 2 년 김태형

1. Vol. 11, Issue 3, 389-404, March 2001 Comparative DNA Sequence Analysis of Mouse and Human Protocadherin Gene Clusters인간과 마우스의 PCDH 유전자 클러스트 들에 대한 DNA서열 비교분석 발표자 석사 2년 김태형

2. Experiment • Designed 19pairsof PCR primers to amplify genomic DNA containing the homologousmouse protocadherin genes • Screen a mouseBAC genomic DNA library (RPCI-23), and isolated 21BAC clonescontaining sequences of the mouse protocadherin gene clusters • Seven minimallyoverlapping clones were selected for DNA sequencing (RPCI-23_193o23,6p18, 72c14, 92d17, 161o8, 56b11, and 19k11) • Comparison of the sequencesof cDNAs with those of the genomic DNA

3. Human Chromosome 5q31 Mouse Chromosome 18 BAC clone reric or psedogene Pcdha variable region exons Pcdhb variable region exons Pcdhc variable region exons C-type Pcdh variable region exons

4. Comparison of the Organization of the Mouse and Human Pcdha GeneClusters • Mouse Pcdha genes confirmed the consensussplice sites at the ends of all 14variable region exons • First 12mouse Pcdha genes are highly similar to eachother • Like the corresponding human genes,mouse Pcdha-C1 and -C2 genes are more similar to each other thanto the 12upstream Pcdha genes • Constant region exons 1,2,and 3are 92%, 99%, and 89% identicalbetween mouse and human

5. Comparison of the Organization of Human and Mouse Pcdhr GeneClusters • 22mouse Pcdhr variable region exons and three small constant region exons in the regiondownstream • The constant region exon sequences are highly conservedbetween mouse and human • Specifically, constant region exons 1,2,and 3have 95%, 90%, and 80% identity, respectively, betweenmouse and human at the nucleotide level • Orthologoushuman gene except the mouse Pcdhr-b8 gene • Mouse has a relic sequenceat the location corresponding to the human Pcdhr-b3 gene • Similarto the Pcdhr gene cluster, the last three Pcdhr genes (C3, C4,and C5) are conserved between mouse and human

6. Comparison of the Organization of Human and Mouse Pcdhb GeneClusters • Single large exon encodes an 818aa protein containing a signal peptide • Highly similar to the human Pcdhb1 protein: 88% identity and 92%similarity with no gaps over the entire length • Covers the gap between thehuman Pcdh8 and Pcdh9 genes, and found that the gap sequencecontains only one additional Pcdhb gene (therefore designatedPcdh8a) • Mouse has six more Pcdhb genes than human does,and the Pcdhb locus is expanded in mouse compared to that in human • The Pcdhb proteins have highly conserved extracellularand transmembrane domains • Conserved Pcdhb 5' splice sites do function. However, neitherthe cell type in which this splicing occurs nor the target 3'splice site has beenidentified

9. Evolutionary Relationships among Members of the Human and Mouse Pcdha,Pcdhb,Pcdhr Genes • Members of Pcdhr genes are strictly conserved between mouse and human • Mouse ortholog of human Pcdhr-b3 gene has degenerated into a relicsequence, and the human ortholog of mouse Pcdhr-b8 has becomea pseudogene • In the sameorder and orientation • C-type protocadheringenes, the last two Pcdha genes and the last three Pcdhr genes,are more similar to each other, and are separated from correspondingupstream genes by a very large intergenic region (>40 kb) in bothmouse and human • Pcdha and Pcdhr gene clusters have highly conserved constant regionexons between mouse and human • Pcdhb gene cluster doesnot have constant region exons in both mouse and human

11. The Distribution of CpG Islands Corresponds to the Locations of the Variable RegionExons • Sequences around the translation start sites of mouse andhuman protocadherin variable region exons revealed a high densityof CpG dinucleotides, suggesting that they are CpG islands • Searched the entire human and mouse gene clusters for CpG islands using the CpGplot program • This distributionsupports the proposal that each variable region exon has its ownpromoter and a transcriptional start site is located upstreamfrom each variable region exon • The peak of ratios correlates with the position of protocadherin variable region exons but not constant region exons

12. Human identity Mouse annotation Mouse genome sequecne

14. Orthologous Human vs Mouse Paralogous

16. Noncoding Sequence Conservation Within the Variable Region of Mouse and Human • Used the PipMaker program (Schwartz et al. 2000) • Systematic analysis of these sequences • First two relics(r1 and r2) in the mouse Pcdha gene cluster • Most striking featuresare the occurrence of highly conserved sequences upstream of eachvariable region exon • 70% identity and longerthan 100base pairs (bp) • 5' flanking sequences of orthologous variable region exons havea significantly higher percentage identity than the correspondingparalogous sequences within Pcdha and Pcdhr gene clusters in bothmouse and human

18. Identification of a DNA Sequence Motif Upstream of Protocadherin Variable RegionExons • Used a version of the Gibbs sampler program called GibbsDNA • This motif cannot be found in transcription factor bindingsite databases • Both human and mouse Pcdhb1genes do not have the motif. • All three gene clusters revealed a common core sequence,“CGCT” • The loci strongly suggest that they are importantfor the regulation of protocadherin geneexpression

19. DISCUSSION • The overall genomic organization of thethree protocadherin gene clusters is highly conserved betweenmouse and human • The interspersedrepeats occupy 41% and 36% of the genomic sequences in the protocadherinloci in mouse and human, 30% in the human T-cell receptor locus • SINEsis much higher than that of LINEs • Identified the orthologous mouseand human gene pairs in the Pcdha and Pcdhr gene clusters

20. METHODS • Mouse BAC Isolation andSequencing • Nineteen PCR primer pairs were designed to screen a mouse BAC library (RPCI-23) • Phylogenetic Analysis PAUP (Phylogenetic Analysis Using Parsimony),version 4.0.0 • Sequence Analysis • Annotation Aligned by using the multiplesequence alignment program Pileup • Comparison RepeatMasker http://ftp.genome.washington.edu/RM/RepeatMasker.html PipMaker http://bio.cse.psu.edu/pipmaker/