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The data from forward-mutation experiments to obtain specific-locus mutations at two closely linked loci in the adenine-3 (ad-3) region of heterokaryon 12 (H-12) of Neurospora crassa have been used to determine the relative frequencies and mutational spectra of ad-3 mutants occurring spontaneously and those induced by 7 different radiation treatments. Previous studies have demonstrated that specific-locus mutants at these two loci result from 5 major genotypic classes, namely two classes of gene/point mutations (ad-3AR and ad-3BR), and 3 classes of multilocus deletion mutations ([ad-3A]IR, [ad-3B]IR and [ad-3A ad-3B]IR). Two different approaches were used to compare spontaneous mutation in the ad-3 region with that induced by 7 different radiation treatments (UV, 32P, 447 MeV protons, 85Sr, 250 kVp X-rays, 39 MeV helium ions, and 101 MeV carbon ions). These comparisons included X2-tests on the numbers of ad-3 mutants resulting in the following two sets of ratios: (1) gene/point mutations and multilocus deletion mutations; and (2) complementing and non-complementing ad-3BR mutants. Combination of the data from these two methods of comparison has demonstrated that each of the 7 radiation treatments induced a spectrum of ad-3 mutants that is statistically different from the spontaneous spectrum. In addition, these same two methods of comparison have been used to compare the mutagenic effects of each of the 7 radiation treatments with each other. Combination of the data from these two methods of comparison demonstrated that the majority of radiation-induced specific-locus mutations: (90.5% (19/21 of the pairwise combinations)) are qualitatively different from each other. We conclude that the mechanisms by which various radiations modify DNA tend to exhibit fundamental differences from each other and from the processes involved in spontaneous mutation.

The data from forward-mutation experiments to obtain specific-locus mutations at 2 closely linked loci in the adenine-3 (ad-3) region of heterokaryon 12 (H-12) of Neurospora crassa have been tabulated to determine the frequency of spontaneous ad-3 mutations and to determine the percentages resulting from each of the 2 major genotypic classes: gene/point mutations and multilocus deletion mutations. Gene/point mutations at the ad-3B locus (ad-3BR) have been characterized to determine the percentage showing allelic complementation to obtain a presumptive identification of the genetic alteration in each mutation at the molecular level. Data from experiments performed at 2 different laboratories have been compared to assess the interlaboratory reproducibility of quantitative data on H-12. No difference was found between the frequencies of spontaneous specific-locus mutations in the ad-3 region. Genetic analysis of 172 ad-3 mutants demonstrated that specific-locus mutations in the ad-3 region result from both gene/point mutations (82.0% [141/172]), and multilocus deletion mutations (14.5% [25/172]). Heterokaryon tests for allelic complementation demonstrated that 52.5% (53/101) spontaneous ad-3BR mutants show allelic complementation, and result from single base-pair alterations. In addition, 100% (25/25) of the spontaneous multilocus deletion mutations result from the 3 smallest sized genotypic subclasses. The implications of the present experimental data for the validation of the ad-3 specific-locus assay system in Neurospora are discussed.

Isoelectrofocusing and high performance liquid chromatographic methods were used to study an abnormal hemoglobin present in a Black male infant and his mother. The variant, named Hb Sinai-Baltimore, focused slightly behind Hb A and separated incompletely from Hb A by cation exchange high performance liquid chromatography, while the separation of the beta A and beta X chains by reversed phase high performance liquid chromatography was complete. The variant was identified through an analysis of peptides in a tryptic digest of the isolated beta X chain and by sequencing of amplified DNA which included the beta-globin gene. The Val->Gly replacement at position beta 18 (codon 18; GTG->GGG) or at the last position of the A helix decreases the stability of the variant without affecting the hematological parameters of its carrier. The propositus was a compound heterozygote for Hb Sinai-Baltimore and Hb S; the relative quantities of the two variant chains were somewhat different from those of the beta X and beta A chains in the mother with the simple Hb Sinai-Baltimore heterozygosity. An uncertainty about the alpha-globin gene status in the child prevented a further evaluation of these differences.

We have identified a severely unstable hemoglobin variant through sequencing of amplified DNA involving the alpha 1-globin gene; the mutation is located in codon 59 (CCG CAG) and results in a Gly-->Asp replacement. This amino acid substitution concerns a glycine residue at an internal position in the E helix, which is in close contact with a glycine residue of the B helix; introduction of the larger and charged aspartic acid residue greatly affects the stability of the molecule. This variant was present in association with a common alpha-thalassemia-1 deletion [-(alpha)20.5 kb] in two adults and caused a severe type of Hb H disease with anemia, low levels of Hb A2, increased zeta chain, and Hb Bart's. In vitro chain synthesis in reticulocytes showed a high specific activity of the variant alpha chain. Only a minute quantity of Hb H was present but instead about 10% of Hb Bart's was observed. The increased synthesis of gamma chains was likely due to specific characteristics of a chromosome with haplotype #3, which was present in both patients. The same family was studied 18 years ago; the improved methodology presently available has led to a corrected diagnosis for these patients.

We have analyzed the hemoglobins of five individual trout from the Ohrid Lake (Salmo L. typicus) by electrophoretic methods, by reversed-phase high-performance liquid chromatography, and by limited structural analyses. The two major classes of hemoglobin are type I (35% of total) and type IV (65%). Type IV is the major oxygen-transporting hemoglobin; it consists of three types of beta chain (in about equal quantities) and three types of alpha chain (one major and two minor types). Several structural differences have been observed between these three beta (IV) chains and between the three alpha (IV) chains, suggesting a complex genetic system governing the synthesis of these proteins. Moreover, a few amino acid substitutions occur at positions involved in contacts between chains, which suggests that differences in oxygen affinity may exist between these various type IV hemoglobins. Type I hemoglobin is less complex because it contains one type of beta chain and two alpha chains; the latter two differ in numerous positions, suggesting duplications of the alpha (I)-globin gene. The alpha and beta chains of type I hemoglobin differ considerably from the alpha and beta chains of type IV hemoglobin, indicating the existence of alpha (I)- and beta (I)-globin genes separate from the alpha (IV)- and beta (IV)-globin genes.

We have identified a new stable abnormal hemoglobin called Hb Valletta, which is characterized by a Thr----Pro substitution at position 87 of the beta chain. This mutation was found to be linked to that of the gamma chain variant Hb F-Malta-I with a His----Arg mutation at position 117 of the G gamma chain. Both variants were detected in the blood samples of 34 Maltese and two Italian newborn babies with isoelectrofocusing and reversed phase high performance liquid chromatography. Similar analyses of cord blood from 388 additional Maltese newborns failed to identify either one of these two variants. Additional analyses of 353 Maltese adults (including 39 beta-thalassemia heterozygotes) resulted in the detection of two adult Hb Valletta heterozygotes. Dot-blot hybridization analyses of amplified DNA with a probe specific for the G gamma-F-Malta-I variant showed that both also carried that mutation. These results show close linkage of the mutant forms of the G gamma- and beta-globin genes, 27-28 kb apart, and a failure to identify chromosomes with either the Hb F-Malta-I mutation alone or with the Hb Valletta mutation alone, indicating a low recombination frequency.

Hb Cleveland is characterized by two amino acid substitutions, namely beta 121(GH4)Glu----Gln as in Hb D-Los Angeles and beta 93(F9)Cys----Arg as in Hb Okazaki, and shares with Hb Okazaki a decreased stability, an increase in oxygen affinity, and decreases in Bohr effect and heme-heme interaction. It is the 13th beta chain variant with two substitutions that has been described thus far.

Hb Davenport is a new, stable alpha chain variant, that was detected in two members of a Caucasian family living in Iowa. Hematological data were within normal limits. It is characterized by an Asn----His replacement at position alpha 78. Mild acidic hydrolysis of the large alpha T-9 peptide, separation of the resulting fragments by reversed phase high performance liquid chromatography, and sequence analysis of a few of these peptides greatly facilitated the identification of this substitution.

Hb Hekinan, an alpha chain variant that is characterized by a Glu----Asp mutation at position alpha 27, was observed in three Chinese females attending a prenatal clinic in Macau. The relative quantities of the stable hemoglobin were 13-14% (average 13.3%); its identification was greatly aided by the separation and purification of the peptides by reversed phase high performance liquid chromatography. Dot-blot analysis of amplified DNA with 32P-labeled probes located the mutation in codon 27 of the minor alpha 1-globin gene; the change involved a GAG (coding for glutamic acid) to GAT (coding for aspartic acid) mutation.

During a survey of cord blood samples of healthy Chinese babies an A gamma variant was detected by its partial separation from normal A gamma in a high performance liquid chromatographic experiment. Structural analysis of the isolated A gamma X chain identified a Val----Met substitution at position A gamma 134; this substitution created a rather unique Met-Met sequence. The ratio of A gamma X to A gamma was about 2 to 3; thus, Hb F with this variant A gamma chain appears to have approximately normal stability.

A severe hemolytic anemia with microcytosis and hypochromia was present in a young adopted Indian patient. Reversed phase high performance liquid chromatographic methodology and heat stability tests detected an unstable alpha chain which was present in 3 to 5% of the total hemoglobin. A larger quantity of the alpha X chain was obtained by preparative reversed phase high performance liquid chromatography. Structural analyses identified an Ala----Pro replacement at position 130 of the alpha chain. The instability of the variant, named Hb Sun Prairie, is comparable to that of Hb Bibba [alpha 136 (H19)Leu----Pro]. Gene mapping failed to detect an alpha-thalassemia deletion (alpha alpha/alpha alpha), while dot-blot analysis of amplified DNA with synthetic probes localized a G----C mutation in codon 130 (resulting in the Ala----Pro mutation) of the alpha 2-globin genes of both chromosomes. These results suggest a homozygosity for the G----C mutation and the condition alpha 2(G----C)alpha 1/alpha 2(G----C)alpha 1 adequately explains the rather severe clinical status of this child, including the marked microcytosis and hypochromia. Unfortunately, family studies to exclude the presence of a large deletion involving all zeta- and alpha-globin genes were not possible.

Three different hemoglobinopathies, i.e. Hb S, Hb Chad [alpha 23 (B4)Glu----Lys], and alpha-thalassemia-2 (-3.7) have been observed in eight members of a family from Surinam. The proposita had all three abnormalities, while her mother and four of her half-brothers had Hb Chad together with an alpha-thalassemia-2 heterozygosity or homozygosity. Gene mapping and dot-blot analysis of amplified DNA identified a G----A mutation in codon 23 of the alpha 2 alpha 1 hybrid gene resulting in the Glu----Lys substitution. The quantity of the alpha-Chad chain averaged 31.5% in its carriers with an additional alpha-thalassemia-2 heterozygosity [-alpha Chad(-3.7 kb)/alpha alpha], and 43% in the two carriers with an additional alpha-thalassemia-2 homozygosity [-alpha Chad (-3.7 kb)/-alpha (3.7 kb)]. These quantities are considerably higher than those reported for families from Chad, China, and Japan; the low levels of 14.5-24% Hb Chad in members of previously reported cases suggest a mutation on a chromosome with two alpha-globin genes [alpha alpha Chad/alpha alpha or alpha Chad alpha/alpha alpha].

The hemoglobins synthesized by the pluripotent K-562 leukemia cell line of human origin after induction with hemin have been isolated by DEAE-cellulose chromatography and characterized by electrophoresis, high pressure liquid chromatography, and a radioimmunological assay. Six hemoglobin zones have been observed with the following likely compositions. Zone 1: alpha 2 epsilon 2, or HB Gower-2; zone 2: zeta 2 epsilon 2, or HB Gower-1; zone 3: zeta 2 gamma 2, or HB Portland-I; zone 4: Hb F, or alpha 2 gamma 2; zone 5: a mixture of acetylated HB Portland-I and Hb F; zone 6: Hb Bart's, or gamma 4. The embryonic Hbs (zones 1, 2, and 3) constituted 50%-75% of the total Hb present; the quantities varied from one experiment to the other. Both Hb Gower-1 and Hb Gower-2 were present. The gamma chain was heterogeneous and contained the G gamma, A gamma I, and A gamma T types in a ratio of about 4:2:1, indicating a heterozygosity for the Ile leads to Thr substitution at position gamma 75. The methodology used can be applied for additional studies evaluating quantitative changes in Hb types due to in vitro manipulations.

A silent beta-chain hemoglobin variant, not detectable by starch gel or agar gel electrophoresis was found using Triton X-100 acid-urea polyacrylamide gel electrophoresis. The abnormal beta-X chain had a more anodic electrophoretic mobility; 38% of the total beta chain was of the abnormal type. Structural analysis using high-performance liquid chromatography and microsequencing procedures indicated a valine to isoleucine substitution at position beta 11(A8). This anomaly did not change the functional properties of the hemoglobin molecule. A mild reticulocytosis was observed in the propositus.

A few rare hemoglobin variants with altered functional properties have been observed in Canadian subjects with either an erythrocytosis or mild anemia. These variants were Hb Alberta [beta 101(G3)Glu----Gly], Hb Linkoping [beta 36(C2)Pro----Thr], a new variant Hb Sunnybrook [beta 36(C2)Pro----Arg], and Hb Caribbean [beta 91(F7)Leu----Arg]. Short clinical descriptions of the subjects are given, the characterization of the variants is described in detail (except for Hb Alberta), while data from some functional analyses are provided. Comparisons with previously published data have been made and the unusual chromatographic property of two abnormal beta chains in a reversed phase high performance liquid chromatographic system is reviewed.

Hb Chicago is a newly discovered hemoglobin variant which was present in a Black newborn baby and her father. The leucine residue at alpha 136, which normally participates in the contact with the heme group, is replaced by a methionine residue. The two heterozygotes were clinically well with normal hematological data. Isolation of the alpha X and alpha A chains by reverse phase high performance liquid chromatography and hydrolysis of these chains with dilute formic acid at 110 degrees C for 24 hours, followed by separation of the resulting peptides by reverse phase high performance liquid chromatography, greatly facilitated the final identification of the abnormality. The baby and both parents had a -G gamma-G gamma-globin gene arrangement on one chromosome (normal: -G gamma-A gamma-) which explains the high G gamma values in the Hb F of these three persons.

A new hemoglobin variant with a Ser----Phe substitution at position beta 49(CD8) was discovered in two members of a family living in the Canary Islands, Spain. Detection was by polyacrylamide gel electrophoresis and by reversed phase high performance liquid chromatography. The variant, which constituted 43% and 45%, respectively, in the two heterozygotes, was slightly unstable. Its presence did not affect hematological values though there was a mild reticulocytosis.

Almost 10 years ago we reported in this journal the characterization of Hb Hacettepe or alpha 2 beta (2)127(H5)Gln----Glu. Unfortunately, we have to conclude that the original characterization of this Turkish variant was in error. The corrected data are presented in this short communication. The variant (alpha 2 beta (2)65(E9)Lys----Met) was (re)named Hb J-Antakya, after the city where the family resides. An abnormal Hb, observed in a Spanish family and named Hb Complutense, had the beta 127 Gln----Glu substitution, erroneously assigned to the Turkish variant.

A new gamma chain variant with an electrophoretic mobility at pH 8.1 between those of Hb S and Hb C was isolated and quantitated by DEAE-cellulose chromatography. It was readily identified with the use of various micro-chromatographic and sequencing procedures as alpha 2 G gamma 2 94(FGl)Asp replaced by Asn. The hemoglobin was named Hb F-Columbus-Ga. The quantity of this G gamma chain variant (as % total gamma chain) was about 39% and the percentages of the normal G gamma and A gamma I chains were 37% and 24%, respectively.