Ung-Jin Kim

Genome sequence of the hyperthermophilic crenarchaeon Pyrobaculum aerophilum

We determined and annotated the complete 2.2-megabase genome sequence of Pyrobaculum aerophilum, a facultatively aerobic nitrate-reducing hyperthermophilic (Topt = 100°C) crenarchaeon. Clues were found suggesting explanations of the organisms surprising intolerance to sulfur, which may aid in the development of methods for genetic studies of the organism. Many interesting features worthy of further genetic studies were revealed. Whole genome computational analysis confirmed experiments showing that P. aerophilum (and perhaps all crenarchaea) lack 5′ untranslated regions in their mRNAs and thus appear not to use a ribosome-binding site (Shine–Dalgarno)-based mechanism for translation initiation at the 5′ end of transcripts. Inspection of the lengths and distribution of mononucleotide repeat-tracts revealed some interesting features. For instance, it was seen that mononucleotide repeat-tracts of Gs (or Cs) are highly unstable, a pattern expected for an organism deficient in mismatch repair. This result, together with an independent study on mutation rates, suggests a “mutator” phenotype.

Mutations in a C. elegans Gqα Gene Disrupt Movement, Egg Laying, and Viability

We find that C. elegans egl-30 encodes a heterotrimeric G protein α subunit more than 80% identical to mammalian Gqα family proteins, and which can function as a Gqα subunit in COS-7 cells. We have identified new egl-30 alleles in a selection for genes involved in the C. elegans acetylcholine response. Two egl-30 alleles specify premature termination of Gqα and are essentially lethal in homozygotes. Animals homozygous for six other egl-30 alleles are viable and fertile, but exhibit delayed egg laying and leave flattened tracks. Overexpression of the wild-type egl-30 gene produces the opposite behavior. Analysis of these mutants suggest that their phenotypes reflect defects in the muscle or neuromuscular junction.

A fosmid-based genomic map and identification of 474 genes of the hyperthermophilic archaeon Pyrobaculum aerophilum

Abstract We have constructed a physical map of the approximately 1.7-Mb genome of the hyperthermophilic archaeon Pyrobaculum aerophilum. Derived from a 12× coverage genomic fosmid library with an average insert size of 36 Kb, the map consists of a single circular contig of 96 overlapping fosmid clones with 211 markers ordered along them. One hundred of the sequence markers have strong similarities to known genes. Many overlaps were also checked using restriction fingerprint analysis. This map is an important step in the elucidation of the sequence of the entire genome of Pyrobaculum aerophilum. To this end we have determined more than 95% of the genome with 15000 random sequences. Each sequence has been screened against the public sequence databases to identify similarities to known genes. We report here a list of the 474 putative genes we have identified.

A human gene similar to Drosophila melanogaster peanut maps to the DiGeorge syndrome region of 22q11

Abstract A Drosophila-related expressed sequence tag (DRES) with sequence similarity to the peanut gene has previously been localized to human chromosome 22q11. We have isolated the cDNA corresponding to this DRES and show that it is a novel member of the family of septin genes, which encode proteins with GTPase activity thought to interact during cytokinesis. The predicted protein has P-loop nucleotide binding and GTPase motifs. The gene, which we call PNUTL1, maps to the region of 22q11.2 frequently deleted in DiGeorge and velo-cardio-facial syndromes and is particularly highly expressed in the brain. The mouse homologue, Pnutl1, maps to MMU16 adding to the growing number of genes from the DiGeorge syndrome region that map to this chromosome.

Final Report: Complete Sequencing of the 2.3Mbp Genome of the Hyperthermophilic Archaeon Pyrbaculum Aerophilum, January 1, 1998 - December 31, 1998

Pyrobaculum aerophilum is a hyperthermophilic archeon discovered from a boiling marine water hole at Maronti Beach, Italy that is capable of growth at 110 C. This microorganism can grow aerobically, unlike most of its thermophilic relatives. Due to the tolerance to oxygen, it is possible to grow this microbe in the presence of air, i.e. on plates. Therefore, it is a good candidate a model organism for studying archaeal biology and thermophilism. Sequencing the entire genome of this organism will provide a wealth of information on the evolutionary and phylogenetic relationship between archaea and other organisms as well as the biology of thermophilism. We have constructed a physical map that covers estimated 2,3 Megabase pair genome using a 10X fosmid library. The map currently consists of 96 overlapping fosmid clones. We have completed sequencing the entire genome using in random shotgun approach with the supplement of oligonucleotide primer directed sequencing. Total 16,098 random sequences corresponding to approximately 3.5X genomic coverage were obtained by sequencing from both ends with vector-specific primers the 2-3 kbp genomic DNA fragments cloned into pUC18/19 vector after shearing have been assembled into a number of contigs using Phrap program developed by Dr. Phil Green at University of Washington, Seattle. Gaps and regions of low quality base calls have been a total of 2,300 directed sequencing and reassembly. Our current full length genomic sequence still suffers from low data quality: only approximately 99% of the nucleotide sequences are accurate. This is mainly due to the low redundancy (3.5 fold) in random sequencing. We plan to perform 2-3,000 more directed sequencing to polish the sequence to 99,99% accuracy. Final polishing of the sequence data and annotation is currently being performed by UCLA team and Caltech sequencing core facility.

Development of BAC libraries and integrated physical mapping of human chromosome 22 using BACs. Annual report, July 1994--June 1995

BACs and fosmids are stable, nonchimeric, and highly representative cloning systems. BACs maintain large-fragment genomic inserts (100 to 300 kb) that are easily prepared for most types of experiments, including DNA sequencing. The authors have improved the methods for generating BACs and developed extensive BAC libraries. They have constructed human BAC libraries with more than 175,000 clones from male fibroblast and sperm, and a mouse BAC library with more than 200,000 clones. The authors are currently expanding human library with the aim of achieving total 50X coverage human genomic library using sperm samples from anonymous donors.