Andrea N. Noyes

Fetal hemoglobin restriction to a few erythrocytes (F cells) in normal human adults

During adult life, the quantity of fetal hemoglobin (HbF) present in F cells--that is, rare erythrocytes which are reactive with rabbit antiserum to human HbF during microscopic immunodiffusion--is sufficient to account for all of the small quantity (less than 0.7 percent) of HbF normally present in whole blood. Thus, erythrocytes are normally heterogeneous with respect to the presence of HbF.

Primate hemoglobins: Some sequences and some proposals concerning the character of evolution and mutation

During a multipurpose survey we examined electrophoretic mobilities of major (A, i.e., α2β2) and minor (A2, i.e., α2δ2) adult hemoglobins from populations of nine primate genera representing a total of 440 New World monkeys and apes. Sequences of hemoglobin chains were inferred from differences in amino acid composition between homologous tryptic peptides supplemented by detailed placement of more than 270 residues. Beta sequences were thus analyzed in five genera (Aotus, Ateles, Hylobates, Saimiri, and Saguinus) and δ sequences in seven (foregoing plus Gorilla and Pan). In most genera, sequences from several individuals, often from several species, were delineated. Fifteen kinds of intraspecies mutants were detected; 10 of these were precisely characterized. Five of the 15 mutants form electrophoretically detected genetic polymorphisms of δ; none such occur in β. Six electrophoretically detected mutants, four in α and two in δ, are uncommon. One of these represents the complete absence of minor component. Three kinds of variants, two in δ and one in β, are electrophoretically neutral and chance findings during sequence analysis of the equivalent of 38 allele products. Two of the neutral variants are not especially common; one may have polymorphic frequency. Several general conclusions stem from these and supplementary findings. First, comparisons of sequences suggest that δ and β genes in all primates either arose from a single event in a common ancestor or from two approximately coincident events. Either assumption allows reconstruction of a reasonably accurate archetype sequence that is effectively common to all descendants. Second, there is a pancellular quantitative disproportion between major and minor hemoglobins ranging from 16:1 to 220:1 in species studied. Delta is consequently presumed to be functionally and adaptively less vital than β. When these premises are adopted, δ is expected to be relatively invisible to natural selection, and, where darwinism is the principal arbiter of evolution and polymorphism, δ is expected to show fewer fixed changes and fewer genetic polymorphisms than β. The opposite is observed. Delta exhibits as many or more changes from archetype than β. This finding and the comparative abundance of δ polymorphism are attributed to nonadaptive factors which are thus considered the source of much evolutionary change. Third, particular sequence positions in various species are the site of recurrent mutations in both β and δ. One such area is occupied by the majority of genetic polymorphisms found in man and other primates. The overall distribution of mutations arising in evolution is remarkably nonrandom in β, δ, and a pool of both. These results are quite unlike most other observations in higher organisms. The sources of such nonrandomness are either selection and/or differential mutability. We rely on our prior assumption of relative selective invisibility for δ and, in part, ascribe the nonrandom distribution of changes to microzones of enhanced mutability. Fourth, the six uncommon electrophoretically detected mutants provide an estimate of heterozygosity (1/73) at hemoglobin loci that is tenfold greater than observed in man. Fifth, the unprecedented chance detection of three kinds of electrophoretically neutral intraspecies mutants among the equivalent of 38 characterized allele products suggests that neutral changes are as common as electrostatically active ones and at least tenfold more common than expected in extrapolation from human variant surveys. Sixth, β analyses from three kinds of gibbon (Hylobates) hemoglobin suggest that one of these is a potentially unchanged relict of the ancient archetype and, further, indicate a degree of homozygous diversity within a species that nearly equals the difference between gibbon and man.

Primate hemoglobins: Polymorphisms and evolutionary patterns

Abstract In this rrport we illustrate the apparently great incidence in the number of different kinds of electrophoretically undetectable intraspecies variants found in the course of characterizing primate hemoglobin α, β and δ sequences. Our primary interest is to assess the significance of such findings in a setting provided by fixed amino acid differences that have accumulated between species during primate evolution. Observed non-randomness in fixed mutations, with respect to both linear distribution and certain kinds of change, is interpreted as due to the restrictive effects of natural selection rather than to either differential mutability at particular positions or disturbed permutations of the genetic code in general. Thus, in the micropatterns of accumulated mutation, we adduce strong evidence for Darwinism. However, we also find that the δ chain of minor hemoglobin A2 (α2δ2)—while apparently visible to selection—seems less restricted than the β chain of the major component, hemoglobin A (α2β2). Such findings are used to support our earlier presumption that comparative changes in β and δ can serve as a model for evaluating mechanisms of amino acid replacement during evolution. We believe that equivalence between β and δ in the numbers of recognizable fixed mutations is more easily explained by non-adaptive mechanisms than by adaptive ones. The frequencies of seven different kinds of electrically neutral variants found within-species during detailed analysis of ∼70 allele products are reported here. Most, if not all, are common. We propose that the overall incidence of different kinds of electrically neutral polymorphisms is probably at least fivefold greater than the kinds of electrophoretically detectable polymorphism. In their electrical neutrality and location the electrophoretically silent polymorphisms strongly resemble the fixed mutations accumulated during evolution of primate hemoglobin chains and are thus candidates for the transient phase of non-adaptive evolution.

Hemoglobins A and A2 in New World Primates: Comparative Variation and Its Evolutionary Implications

Hemoglobin A2 (α2δ2) in New World primates represents about 1/160 to 1/16 of total hemoglobin and, by virtue of this low proportion, is presumed to be functionally unimportant. Nonetheless, A2 exhibits genetic polymorphism by electrophoresis in three out of five genera, whereas the major component, hemoglobin A (α2β2), is electrophoretically invariant. Moreover, in four genera, including man, the evolutionary accumulation of mutations has been greater in δ than in β Such findings suggest that both polymorphism and evolutionary changes can accrue to an effectively functionless and thus selectively nearly netutral gene.

Silent hemoglobin alpha genes in apes: potential source of thalassemia.

Small quantities of unusual hemoglobins were found in 1 of 37 chimpanzees and 2 of 6 gorillas. In each genus these hemoglobins contain unique α chains that differ from the ordinary by eight to nine scattered amino acid changes. The unusual chains arise from a hitherto undetected hemoglobin 3α locus. No 3α products are found in most apes; accordingly, 3α is considered synthetically inactive in all but a few reversion mutants. Indirect evidence that the inactive 3α locus is juxtaposed to an active α locus together with the supposition that 3α exists in man provides a setting wherein thalassemia might be produced by nonhomologous recombination between two loci.

Developmental changes in human erythrocyte carbonic anhydrase levels: Coordinate expression with adult hemoglobin

In order to bolster the argument that parallel developmental changes in erythrocyte adult hemoglobin (HbA) and carbonic anhydrase (CA) content provide a potentially suitable model for the dissection of coordinate gene expression, the magnitude of fetal vs adult differences in CA I and CA II levels was examined in human red cell subpopulations obtained after varying periods of exposure to CA-dependent, NH4Cl-HCO-3-mediated, acetazolamide-modulated hemolysis. When content of CA I and CA II was immunologically assessed in cohorts surviving successively longer periods of hemolysis, cord blood red cells were divisible into two populations. Fifteen to thirty percent are rapidly disrupted and have CA I and CA II concentrations similar to those in adult blood erythrocytes. The remaining 70 to 85% have CA I concentrations which are 100-fold less and CA II concentrations which are 5- to 20-fold less than those found in adults. Thus, contrary to past reports, the magnitude of the developmental change in CA I concentration closely resembles the magnitude of change in HbA levels.

IMMUNOLOGICAL BASIS FOR DETECTION OFSICKLE CELL HEMOGLOBIN PHENOTYPESIN AMNIOTIC FLUID ERYTHROCYTES

In attempting to develop a benign procedure for ascertaining adult hemoglobin A, AS, and S phenotypes during early fetal life we adopt the viewpoint that-in terms of risk-amniotic fluid is the only presently acceptable source of cells for typing a live fetus. With this self-imposed restriction in mind and lacking a means for inducing adult hemoglobin synthesis in nonerythrocytic cells, we have relied on microscopic immunodiffusion analysis of single erythrocytes. This technique, developed by Gitlin and colleagues and independently by Daufi and Rondell,? depends on the formation of pericellular spheroids of immunoprecipitate granules. Granular spheroids develop as agar-mounted erythrocytes break and release their antigenic hemoglobin contents into an antibody-containing medium. Spheroid size is a function of the amount of soluble antigen present and thus assay is roughly quantitative. The procedure lends itself, appropriately for our purpose>, to detecting quantities of hemoglobin which represent as little as -1/400 of adult erythrocyte content, i.e., -700,000 hemoglobin molecules 1 attomole = 80 femtograms. We have previously attained this level of sensitivity in assaying the quantity of hemoglobin produced by certain mouse leukemic cells.‘ In this report we describe our procedures, the details and requirements of our strategy, results to date, and some features of uncommon fetal hemoglobin bearing erythrocytes (denoted “F-cells”) which occur in the peripheral blood of all adults examined. We emphasize that our studies have not yet progressed to the point of actually typing hemoglobin A and S in the unborn. We are, however, easily able to detect adult hemoglobin in cells from individuals 13-19 weeks gestational age.

THE STRUCTURE AND BIOSYNTHESIS OF HEMOGLOBINS A AND A2 IN THE NEW WORLD PRIMATE Ateles PANISCUS: A PRELIMINARY ACCOUNT*

Variations in the quantities of structurally different hemoglobins in man provide several natural models for the dissection of genetic regulatory mechanisms. Among the kinds of variation that exist are: (1) the 40:l disproportion in concentration between hemoglobins A (cu~(32) and A2 ( ~ ~ 2 6 2 ) ; (2) the numerous inequalities between the amounts of A and mutant hemoglobins in heterozygotes such as A/S individuals; (3) the switch from fetal to adult hemoglobins in the neonatal period. In principle, variations such as these should offer unexcelled opportunities for delineating the various mechanisms responsible for the quantitative regulation of proteins in differentiated organisms. In practice, however, the usefulness of such models is severely limited-even in moderately anemic subjects-by the poverty of realizable incorporation of radioactive amino acids into hemoglobin chains. For example, recent investigations of the patterns of in vitro synthesis of nascent (3 and 6 polypeptide chains in the reticulocytes of human subjects have been somewhat handicapped by vanishingly small radioactive counts. Accordingly, supplementary models have been sought in experimental animals wherein adequate anemia and biosynthesis can be realized. Several model systems have been described including the anemia dependent switch of hemoglobins in ~ h e e p , ” ~ ~ goats and, more recently and more modestly, in ducks. Another kind of model-similar perhaps to the A/mutant hemoglobin disparity in man-is provided by natural inequalities in the amounts of several adult hemoglobins in certain Old World monkeys’ and domestic cats.” In the present paper, discussion is confined to the model afforded by the 17:l ratio in the relative proportions of hemoglobins A and A2 in Ateles paniscus, a New World spider monkey. Although the existence of an Az-like hemoglobin in New World primates (but not in other primates aside from apes and ourselves) has been known” since 1957, 1

IMMUNOLOGICAL PURIFICATION AND CHARACTERIZATION OF HEMOGLOBIN CHAIN‐SYNTHESIZING POLYSOMES: A DERIVATIVE HYPOTHESIS FOR REGULATORY AND EVOLUTIONARY INTERRELATIONSHIPS IN HEMOGLOBIN BIOSYNTHESIS *

Immunological isolation of mRNA-specific polysomes proceeds on the now rigorously proved assumption *-( that antibodies directed against a particular polypeptide chain bind to forms of that chain that are still growing and attached to polysomes. Polysomes coated with specific antibodies to chain can, in turn, be selectively precipitated either by completing the immunological reaction with appropriate amounts of antigen, as used for antiovalbumin bound to hen oviduct p01ysomes,~-~ or, as described here, through use of antiantibody (e.g., rabbitanti-goat-IgG reactive with goat-anti-rabbit-hemoglobin fl chain) in the case of antihemoglobin chains bound to reticulocyte poly~omes.~ In the latter instance use of anti-antibodies is a necessity since some of the most useful antibodies produced against hemoglobin chains are of a nonprecipitating kind. This is, for example, true of goat-anti-rabbit-fl chain produced by each of 6 animals and goat-anti-human-y chain produced by 2; all of these reagents are entirely nonprecipitating when purified. The resulting need for anti-antibodies is not, however, without advantages. It may allow a more sparing use of sometimes limited quantities of antibodies than is possible with procedures that depend on carrier antigen for precipitation. Furthermore, our experience with antiantibodies in reticulocyte polysome isolation can perhaps later be transferred to situations where antigen itself is in short supply and unavailable for completion of an immune reaction. In this report, we first describe some of the requisites for immunological purification of hemoglobin chain-specific reticulocyte polysomes and second, give an immunological characterization of these polysomes. We use the latter to develop an overall view of some of the evolutionary and regulatory interrelationships involved in hemoglobin biosynthesis. In particular, we propose that ubiquitous a gene duplication is the source of the considerable excess of u mRNA.