Hemoglobin

Based on knowledge of other mammalian species, Peromyscus undoubtly has a compound locus which produces beta like chains, and an independent locus which produces alpha like chains. Electrophoresis of Peromyscus whole hemoglobin, or dissociated chains produce an array of phenotypes which have been variously interpreted. This array includes includes variations in the number of electrophoretic bands and quantitative differences in the intensites of the electromorphs. In mature Peromyscus most, but not all, species typically have two whole hemeoglobin electromorphs of more or less equal intensity, generally refered to as the "normal" or "double-banned" phenotype (Foreman, 1968). This is generally true of P. maniculatus, but some populations have "single-banned" or "triple-banned" individuals. How hemoglobin genetics of P. maniculatus is related to hemoglobin variation in other species in the genus is discussed in Sections 6 and 8.

The formal genetics of hemoglobin in P. maniculatus is confused by several factors: (1) It has not yet firmly been established whether genetic variation involves the beta - like locus, the alpha - like locus or both. (2) Quantitative differences, as opposed to discrete electrophoretic patterns, may be due to dosage effects at single loci, or to independently inherited regulator genes. (3) The available information is interpretable by different hypotheses. (4) Tightly linked site duplications, where there is a variant at one site, will segregate essentially as alleles at a single locus, thus two or more site duplications in tandem would be interpreted by formal genetics as a single locus, but by molecular genetics as two or more loci. (5) Investigators have used different nomenclature for "alleles" and "loci". In general the nomenclature established by Rasmussen, et al. (1968) differs from that of Snyder (1978), and neither is completely consistent with that used for humans or laboratory mice.

Considering these difficulties, for the purposes of this review, it is assumed that there are two independently inherited compound loci, alpha - like and beta - like. Both loci have tandem tightly linked sites, each of which code for an intact hemoglobin chain. Further assumptions are that within any site of a compound locus a mutation may occur which could potentially alter the electrophoretic mobility, yielding a genetic variant. This variant would behave genetically as an allele at the compound locus. Although the principle of "one gene - one polypeptide product" appears to be violated because of the improbability of recombination within a compound locus, it is understood that from the molecular aspect, each "site" is, in the strict sense, a "locus".

Rasmussen, et al. (1968) analyzed genetic variation in the more anodal electromorph of whole hemoglobin. The slow electromorph was invariant. They referred to the fast electromorph with the Roman numeral I and symbolized the locus HbI, since it could not be determined from their data whether the alpha - like or beta - like locus was involved. Snyder (1978) analyzed discrete and quantitative variation by electrophoresis and densitometry of whole hemoglobin, as well as separated globin chains. Additionally, he employed molecular hybridiaztion. He designated three variant loci, Hba, Hbb, and Hbc, together with two monomorphic loci Hbd and Hbe. Hba, and Hbc are alpha - like whereas Hbb, Hbd, and Hbe are beta - like. Maybank and Dawson (1976) described variation in the more anodal electromorph of both whole hemoglobin and globin chains. The later concluded, based on the anodal mobility of the separated chains, that the HbI locus of Rasmussen, et al. (1968) was probably beta - like. However, the HbI locus is independent of the c locus in P. maniculatus (Maybank and Dawson, 1976), whereas the beta - like locus of both Mus and Rattus are linked to the c locus by approximately 5 centimorgans. Fruther, Snyder (1978) hypothesized that the HbI^f variation of Maybank and Dawson (1976) was at the alpha - like locus, and Jensen, et al. (1976) proposed a model for HbI variants known to occur. Although their model was demonstrated using beta - chain variants, they acknowledged that eigher chain could be involved.

Hemoglobin phenotypes are determined by a variety of electrophoretic and densitometric procedures. Washed erythrocytes from adult animals are lysed in a buffer. Starch, polyacrylamide and cellulose acetate gels have all been used with a variaty of buffer systems, generally at a pH about 8.5. Hemoglobin is the major protein in the system and may be observed directly on the gel without staining, but more usually is stained with amido black general protein stain. Intact hemoglobin migrates anodally at this pH.

Five electromorphs (clectrophoretic bands) for intact hemoglobin are known in Peromyscus (Foreman, 1968; Snyder, 1978). Four of these occur in P. maniculatus, but, as previously noted, were variously designated by different investigators. Snyder (1978) has assigned numbers to the electromorphs in decreasing order of mobility, 1 through 5. Electromorph Hbl occurs in P. truei, P. califomicus and P. gossypinus, but is not known in the P. maniculatus group. The table below gives the synonomy of the electromorphs in P. maniculatus. Although Snyder (1978) lists twelve phenotypes, certain ones are not clearly distinguishable. Ten phenotypes can be resolved involving major quantitative distinctions in the Hb 2, 3 and 5 electromorphs, which may be "faint", "intense" or absent. No individual P. maniculatus has been known to have more than three electromorphs of adult hemoglobin.

The globin chains may be separated from the intact tetramer using acetic acetone and 6 to 8 molar urea in a variaty of procedures. The extracted globin chains are electrophoresed on cellulose acetate or polyacrylamide and visualized with amido black. By analogy with human and other globins, the beta - like globins migrate anodally, whereas the alpha - like globins migrate cathodally or remain at the orgin. Three anodally migrating globins phenotypes were distinguished by Maybank and Dawson (1976) and six cathodally migrating phenotypes were recognized by Snyder (1978).

A distinct fetal hemoglobin is produced in P. maniculatus (Maybank and Dawson, 1976), which, by analogy with other myomorphic rodents is the product of the d site of the b - like compound locus. Adult hemoglobin appears by the eighteenth day of gestation, presumably by the production of b chains by the b site, and the d site ceases to function. Peromyscus fetuses at 15 days gestation have two electromorphs, the Hb 4 or S plus the distinct more slowly migrating fetal band. The fetal hemoglobin may corespond to the faint Hb 5 described by Snyder (1978).

The species P. polionotus (Biggers and Dawson, 1971) and P. melanotis (Bowers et aL, 1973) have been surveyed for hemoglobin polymorphisms. Both have the common "double-band" phenotype of most P. maniculatus. Foreman (1964) reported that the tryptic peptide digest of hemoglobin from P. maniculatus differ from that of P. polinotus in two peptides, thus the hemoglobins of these two species are not identical, despite their similar electrophoretic mobilities at pH 8.6. A slight difference in whole hemglobin electrophoretic between semi-domesticated laboratory stocks of P. maniculatus (BW stocks) and P. polionotus (PO stock) is noted at lower pH. (Shaffer, unpub.)

ALLELES: Hbd⁰, Hbd¹, Hbd²
Hbe⁰ (hypothetical), Hbe¹

SYNONOMYS: Hbd⁰ = Hbl⁰
Hbd¹ = Hbl^a = Hbl^r
Hbd²= (Hbc²) =Hbd^f
Hbe¹ ? = Hb (fetal)

LINKAGE: Linkage Group I (Chromosome 1 ?)

DNA SEQUENCE: Genbank Accession #s M15292, M15289, M15294, M15297, M15293, M15290, M15295, M15298, M15291, M15296, M15299. Partial sequences, tandem genes Hbb-bl, b2, b3.

PHENOTYPES: Phenotypes are based on electrophoresis of lysed erythrocytes.

ALLELES: Hbc⁰, Hbc¹

SYNONOMY: Hbc^c = HbI⁰

PHENOTYPES: Phenotypes are based on electrophoresis of lysed erythrocytes.

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