Kenneth James Livak

The world-wide distribution of allele frequencies at the human dopamine D4 receptor locus.

Abstract The dopamine D4 receptor gene (DRD4) has an expressed polymorphism in the third exon that may have functional relevance. The polymorphism exists at two levels. At the higher level there is an imperfect tandem repeat of 48 base pairs (bp) coding for 16 amino acids; alleles have been identified with 2 (32 amino acids) to 10 (160 amino acids) repeats. The imperfect nature of the repeats is responsible for a more subtle level of variation since alleles with the same number of repeats can differ in the exact sequences or in the order of the variants of the 48-bp unit. We have undertaken a global survey of this expressed polymorphism as one approach to understanding the evolutionary significance and origins of the polymorphism as well as understanding what selective forces, if any, may be operating at this locus. As the first step, we have determined the repeat number genotype of the DRD4 repeat polymorphism in 1,327 individuals from 36 different populations. The allele frequencies differ considerably among the different populations. The 4-repeat allele was the most prevalent (global mean allele frequency = 64.3%) and appeared in every population with a frequency ranging from 0.16 to 0.96. The 7-repeat allele was the second most common (global mean = 20.6%), appearing quite frequently in the Americas (mean frequency = 48.3%) but only occasionally in East and South Asia (mean frequency = 1.9%). The 2-repeat allele was the third most common (global mean frequency = 8.2%) and was quite frequent in East and South Asia (mean frequency = 18.1%) while uncommon in the Americas (mean frequency = 2.9%) and Africa (mean frequency = 1.7%). The universality of the polymorphism with only three common repeat-number alleles (4, 7, and 2) indicates that the polymorphism is ancient and arose before the global dispersion of modern humans. The diversity of actual allele frequencies for this expressed polymorphism among different populations emphasizes the importance of population considerations in the design and interpretation of any association studies carried out with this polymorphism.

The effects of hybrid ribosome-binding-site variants on the expression of human Interferon-β in Escherichia coli

Human beta-interferon (IFN-beta) has been expressed in Escherichia coli by using vectors that join the trp promoter and a hybrid ribosome-binding site (HRBS) to the mature IFN-beta coding sequence. Introduction of an NcoI site at the ATG initiation codon of IFN-beta by site-directed mutagenesis has facilitated the construction of a series of portable HRBSs, by utilizing oligodeoxynucleotide adapters. The spacing between the Shine-Dalgarno (S-D) sequence and the initiator ATG ranged from 7-13 bp. One spacer of 9 bp length was varied at a single position. IFN-beta expression by these HRBS variants, as analyzed by antiviral activity and SDS-PAGE, shows both nucleotide sequence and spacer length effects. In vitro transcription-translation experiments indicate that some single base changes in the HRBS significantly decrease the rate of translation of the IFN-beta mRNA.

Polymorphisms in the human DNA ligase I gene (LIG1) including a complex GT repeat

Sequencing of a human DNA ligase I cDNA clone derived from HeLa cells revealed two unreported differences with the published sequence: a single base change and a three-base deletion. Both differences are in exon 6, and were analyzed by amplifying a segment containing exon 5, intron 6, and exon 6. The first finding was that intron 6 is approximately 2.6 kb in size, not the 1 kb reported in the literature. By sequence analysis of amplified segments, the single-base difference in exon 6 was shown to be polymorphic, with HeLa cells heterozygous for the A/C difference. Analysis of 60 unrelated individuals found a frequency of 0.5 for each allele. Primer extension reactions across the exon 5/exon 6 boundary were performed on cDNA obtained from HeLa cells and human thymus. The results show that the three-base deletion is due to a variation in splicing. For both HeLa and thymus, two-thirds of the transcripts are like the published cDNA sequence and one-third have the three-base deletion. Finally, sequencing of part of intron 6 revealed the presence of a complex GT repeat consisting of a 48-50 nucleotide polypurine tract followed by a variable number of GT residues. This entire unit of polypurine tract plus GTs is repeated three times. Detection of the repeated sequences required the development of specialized cloning and PCR conditions. Analysis of a pedigree showed that this complex repeat is polymorphic.