Comparative Analysis of Human Chromosome 22q11.1-q12.3 with Syntenic Regions in the Chimpanzee, Baboon, Bovine, Mouse, Pufferfish and Zebrafish Genomes. Dr. Bruce A. Roe George Lynn Cross Research Professor Advanced Center for Genome Technology Department of Chemistry and Biochemistry
Comparative Analysis of Human Chromosome 22q11.1-q12.3 with Syntenic Regions in the Chimpanzee, Baboon, Bovine, Mouse, Pufferfish and Zebrafish Genomes
Dr. Bruce A. Roe
George Lynn Cross Research Professor
Advanced Center for Genome Technology
Department of Chemistry and Biochemistry
University of Oklahoma
LXVIII CSHL Symposium
“The Genome of Homo Sapiens”
May 28 - June 3, 2003
“The joy of science is the people you meet along the way and how they influence your life”
Jochanan Stenesh and Lilian Myers at Western Michigan University
and Bernie Dudock at SUNY Stony Brook
Bart Barrell and Alan Coulson originally at the MRC-Hills Road Cambridge and Ian Dunham both now at the Sanger Institute
Bev Emanuel at Childrens Hospital of Philadelphia
Watson and Crick
Sanger, Keio, Wash U, OU
Human Chromosome 22 Sequence Features
*Shoemaker DD., et al. Experimental annotation of the human genome using microarray technology. Nature. 409, 922-7 (2001).
Siblings by 1 to 2 million bases, ~99.98% identical, with coding regions 99.99999% identical
Unrelated humans by 6 million bases, ~99.8% identical overall, with coding regions 99.9999% identical
Chimpanzees by about 100 million base pairs ~98% identical
Baboons by about 300 million base pairs ~92% identical
Mice by about 2.8 billion bases, but coding regions are ~90% identical
Leaf spinach by about 2.9 billion bases, but coding regions are ~40% identical
Differences between individuals
The yellow underlined sequence is the first exon of the BCR gene involved in leukemia. Only 5 bases (N) differ in non-gene regions.
Human Chromosome 22
Single Nucleotide Polymorphisms*
Number of overlaps335
Size of overlaps13,203,147 bp
Number of SNPs11,116 (~1/1000 bp)
Number of substitutions9,123 (82%)
Number of ins/del1,193 (18%)
Only 48 of the 11,116 SNPs were in coding regions ~ 10 fold lower than in non-coding
*E. Dawson, et al. A SNP Resource For Human Chromosome 22: Extracting Dense Clusters of SNPs from the Genomic Sequence. Genome Research, 11, 170-178 (2001).
“We each are like a different symphony orchestra”
“All playing the same instruments slightly differently”
Where we stand now
Chimpanzee and Baboon Genomic Sequencing
Questionable gene present in primates but not in rodents
PIP Plot of a region of human chr22 compared to syntenic regions of baboon and mouse
Variations in the regions syntenic to the human chr 22 immunoglobulin light chain region from chimp, baboon, rat and mouse
34 Kbp deletion in baboon
Exons in one copy of a zebrafish duplicated gene with 75% homology to human but greatly diverged, <50% homology, in the other copy
Instance of a rare alu deletion in chimp and a gene having very low homology in fish
Conclusions from the analysis of vertebrate genomic sequences
“Zebrafish are small people that swim in the water and breathe through gills”
Han Wang, Dept. Zoology and Director of the University of Oklahoma Zebrafish Facility
How much of the ~1.7 Gbp genome has been sequenced so far?
The whole genome shotgun project comprises roughly 11.6 million traces by now. With an average quality clipped trace length of 517 bp this adds to 6 Gb in total, so the genome is covered 3.5 times.
The new assembly Zv2 is built on 11.7 million traces with an average trace length of 651 bp length, adding up to 7.64 Gbp (4.5 x coverage).
The current Sanger Institute in-house statistics for the clone sequencing are:
* 322,712,747 bp unfinished
* 112,494,895 bp finished
* 435,207,642 bp total
Zebrafish Developmental stages(HPF*)
Zygote Period (0-3/4 h)
The newly fertilized egg is in the zygote period until the first cleavage occurs
Cleavage Period (0.7- 2.2 h)
After the first cleavage, blastomeres divide at approximately 15 minute intervals
Blastula Period (2 1/4 - 5 1/4 h)
Begins at 128-cell stage or 8th zygotic cell cycle. Embryo enters midblastula transition (MBT), the onset of zygotic transcription. Period ends at the onset ofgastrulation.
Gastrula Period (5 1/4 - 10 1/3h)
Morphogenetic cell movements of involution, convergence, and extension occur, producing the primary germ layers and the embryonic axis.
Segmentation Period (10 1/3 - 24 h)
Somites develop, the rudiments of the primary organs become visible, the tail bud becomes more prominent and the embryo elongates. The first cells differentiate morphologically, and the first body movements appear.
Pharyngula Period (24-48 h)
Embryos developing to the phyolotypic stage when it posesses the classic vertebrate bauplan.Migration of the posterior lateral line primordium. Rapid organogenesis continues.
Hatching Period (48-72 h)
Individuals within a single developing clutch hatch sporadically during the whole period.
Kimmel CB, et al. Stages of embryonic development of the zebrafish. Dev Dyn 203, 253-310 (1995).
Gene Expression in Zebrafish
Gene Expression in Zebrafish (cont)
Probe1 b6 shows hybridization in the brain from 24 hours onward and in the eye from 48 hours onward.
1b6: AP000557.1.mRNA chr22 position:18495442-18504448 KIAA1020 hypothetical protein matches EST b6n20zf
Whole mount in situ hybridization with ssDNA-digoxigenin labeled probe made from a PCR product. Brain-specific expression of this mRNA during embryonic development
Anti-sense probe Sense probe No probe
The importance of a “no probe” antibody staining control to determine if any probe-independent antibody staining occurs in the lens
Typically only see anti-sense probe hybridizing, and therefore stained by anti-dig antibody with some probe-independent staining in the eye.
72 hour post fertilization embryo
A probe to the unique 3’ UTR if there are multiple paralogs
One last experiment with a surprise ending
Hybridization probe a8h24
unique to 3’ UTR of zebrafish gene 2 based on our zebrafish EST sequence
Anti-sense probe Sense probe No probe
One too many controls sometimes results in a surprise observation
Both the anti-sense and sense probes hybridized to 72 hour post fertilization embryonic brain.
Indicating RNA transcribed from the opposite, non-coding strand?
Mary Catherine Williams
Work-study Undergraduate students**
Bruce Roe, PI
Pheobe Loh *
* Previous undergraduate res. student
** Present undergraduate res. student
*** Previous graduate student
**** Present graduate student
Funding from the NHGRI, Noble Foundation, DOE, NSF (pending) - Collaborators at Sanger, CWRU, CHOP, Keio, UIUC and Riken
Peggy and Charles Stephenson Center