Oliver Smithies

A comprehensive set of sequence analysis programs for the VAX

The University of Wisconsin Genetics Computer Group (UWGCG) has been organized to develop computational tools for the analysis and publication of biological sequence data. A group of programs that will interact with each other has been developed for the Digital Equipment Corporation VAX computer using the VMS operating system. The programs available and the conditions for transfer are described.

The structure and evolution of the human β-globin gene family

Argiris Efstratiadis Department of Biological Chemistry Harvard Medical School Boston, Massachusetts 02115 James W. Posakony, Tom Maniatis, Richard M. Lawn* and Catherine O’Connell+ Division of Biology California Institute of Technology Pasadena, California 91125 Richard A. Spritz, Jon K. DeRiel,# Bernard G. Forget and Sherman M. Weissman Departments of Genetics and Internal Medicine Yale University School of Medicine New Haven, Connecticut 06510 Jerry L. Slightom, Ann E. Blechl and Oliver Smithies Laboratory of Genetics University of Wisconsin Madison, Wisconsin 53706 Francisco E. Baralle, Carol C. Shoulders and Nicholas J. ProudfootQ MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 2QH, England Summary We present the results of a detailed comparison of the primary structure of human p-like globin genes and their flanking sequences. Among the se- quences located 5’ to these genes are two highly conserved regions which include the sequences ATA and CCAAT located 31 2 1 and 77 + 10 bp, respectively, 5’ to the mRNA capping site. Similar sequences are found in the corresponding locations in most other eucaryotic structural genes. Calcula- tion of the divergence times of individual @like globin gene pairs provides the first description of the evolutionary relationships within a gene family based entirely on direct nucleotide sequence com- parisons. In addition, the evolutionary relationship of the embryonic e-globin gene to the other human P-like globin genes is defined for the first time. Finally, we describe a model for the involvement of short direct repeat sequences in the generation of deletions in the noncoding and coding regions of B-like globin genes during evolution.

A Simple Hemagglutination System Requiring Small Amounts of Red Cells and Antibodies

A method is described which permits dilute red cell suspensions (1/32%) to be used in plastic microtiter plates for detecting hemagglutinins at unusually high dilutions. The use of suitable additives to the hemagglutination mixture enables excellent settling patterns and a high sensitivity to be obtained. Direct agglutination, inhibition, enzyme promoted agglutination, and Coombs tests can all be performed. Quantitative studies demonstrate the discriminatory powers of the technic. The method may be useful for economizing in the consumption of reagents in blood grouping.

A history of the human fetal globin gene duplication

The nucleotide sequence of 11.4 kilobase pairs (kb) of human DNA that includes the two fetal globin genes, G gamma and A gamma, shows that they are part of a 5 kb tandem duplication. A small segment of DNA occurs three times, on either side of and between the two duplicates. We present two models that account for these observations. One model is simple but requires the assumption of a preexisting repetitive element; the other is more complex but does not require the assumption of preexisting repeats. Over much of the 5 kb duplicated region, the present duplicate copies differ by an average of 14% of their bases, from which we calculate that the duplication was first formed about 34 million years ago. However, 1.5 kb of DNA are vitually identical in the two genes analyzed here, probably as a consequence of an intergenic exchange (gene conversion) that replaced part of the diverging A gamma gene with the corresponding part of the G gamma gene. This conversion took place around 1 million years ago. A sequence of repeated dinucleotides may be one signal involved in exchanges leading to this and similar conversions. Cycles of duplication, triplication, deletion and gene conversion on probably common in many multigene families.

Initiation of protein synthesis at an unusual position in an immunoglobulin gene

The amino acid sequence of urinary β2-microglobulin has been partially determined and found to be related to the constant region of IgG immunoglobulin heavy chain. β2-Microglobulin is present in normal individuals. Its gene may have evolved from an immunoglobulin gene by the use of an unusually located start signal for initiating synthesis of the polypeptide.

Lactic Dehydrogenase: Genetic Control in Mana

A genetically determined variant of lactic dehydrogenase has been observed in the red cells of four members of two generations of a Brazilian family. The appearance of the variant after starch-gel electrophoresis of the hemolysates supports the concept that the lactic dehydrogenase isozymes are determined by the interaction of two subunits which are under separate genetic control. The products of the mutant and normal allele do not, however, appear to associate randomly to form isozymes. The similarity of the relative retardation of normal and mutant isozymes in gels made with increasing concentrations of starch suggests that they do not differ significantly in size.

Disulfide-bond cleavage and formation in proteins.

Disulfide bonds can be cleaved at an alkaline pH by treating a protein with excess of a reagent disulfide in the presence of catalytic amounts of thiol. The cleavage products are stable and can be isolated; they contain the mixed disulfide between the reagent and the exposed thiol groups of the protein. The extent of cleavage is readily controlled by the pH of the reaction, temperature, and the addition of urea. Disulfide bonds cleaved by the reaction can be re-formed by exposing the mixed disulfide of the protein to catalytic amounts of thiol. Specific side chains can be added on to the thiol groups in native proteins by treatment with a reagent disulfide alone.

Genetic Polymorphism of C'3(β1c-Globuiin) in Human Serum

Genetic polymorphism of the third component of human complement and its breakdown products has been detected in human serum by high-voltage starch-gel electrophoresis. Six phenotypes were observed in a study of 113 randomly chosen Caucasians. Their inheritance is controlled by four codominant alleles at an autosomal locus. The gene frequencies in this study were C31, 0.21; C32, 0.77; C33, ∼0.01; and C34, ∼0.004.

Antibody Variability Somatic recombination between the elements of "antibody gene pairs" may explain antibody variability

I have analyzed the available amino acid sequence data from 30 myelomatosis-derived proteins. Several types of variation are apparent. I conclude that a major and genetically predetermined contribution to the variability of these proteins and of antibodies could be provided by chromosomal rearrangements resulting from somatic recombination between similar but not identical genes in antibody gene pairs. My hypothesis suggests many new types of experiment and can be tested (31).

Base substitutions, length differences and DNA strand asymmetries in the human Gγ and Aγ fetal globin gene region

Abstract We have studied differences arising subsequent to the 5 kilobase pair (kb) duplication that led to the human G γ and A γ fetal globin genes. The local occurrence of base substitutions in the duplicated 5 kb region correlates positively with the local AT base pair content. This correlation also occurs in two mouse β-globin genes and in two mouse immunoglobulin genes. The relationship is valid for transcribed or nontranscribed DNA and for DNA that contains only coding sequences. Length differences in the fetal globin duplicated regions correlate positively with the occurrence of short direct repeats of ≥5 base pairs. Path analysis of the interrelationships of base composition, base substitutions, repeats and length differences provides an integrated view of the relative effects on chromosomal changes of these variables and of selection. The distributions along the chromosome of simple sequences and of base compositions show highly significant local asymmetries between the transcribed and nontranscribed strands of the DNA, which permit us to divide the fetal globin gene region into chromosomal domains. Comparable domains are present in DNA from other sources, including the mammalian viruses SV40 and polyoma virus strain A-2 in which some of the domains appear related to discrete functions.