Studies on the genetic polymorphism of lactate dehydrogenase B (phenotype B−) in rodent erythrocytes

While the common red cell phenotype of lactate dehydrogenase in mammals includes A and B subunits, in some species, especially of the rodent suborder Myomorpha, LDH B subunits are absent in erythrocytes (phenotype B^–). A polymorphism involving LDH B⁺ and LDH B^– phenotypes was observed in Mus musculus, and it was found that LDH B^– is recessive in relation to LDH B⁺ (Shows and Ruddle, 1968). A similar polymorphism is described here in the field mouse, Apodemus sylvaticus. In family studies, the recessive mode of inheritance is confirmed. Induction of reticulocytosis reveals that the LDH B^– phenotype is not the consequence of degradation of the B subunits in aging red cells. The nature of the mutation giving rise to this polymorphism is discussed.Supported by the Deutsche Forschungsgemeinschaft.     

Postnatal changes in canine erythrocyte pyruvate kinase isozymes

The isozyme pattern of pyruvate kinase in canine erythrocytes changes following birth. These changes have been followed by electrophoretic, immunologic, and kinetic measurements of the isozymes. At birth, a mixture of isozymes is present consisting of the M₂ isozyme and hybrid molecules containing M₂ and R subunits. With increasing animal age, the content of M₂ subunits decreases and the content of R subunits increases. At 6 months of age, the isozyme pattern is indistinguishable from that of adult erythrocytes which contain only the R tetramer. We conclude that there is a switch in erythrocyte pyruvate kinase gene expression during the first 6 months of postnatal life. The existence of hybrid molecules during the switch indicates that both M₂ and R genes are expressed within each erythroid precursor cell. The developmental changes in erythrocyte pyruvate kinase are consistent with the role of this enzyme in the regulation of the oxygen-transport function of canine hemoglobin by 2,3-diphosphoglycerate in the postnatal period.This research was supported by Public Health Service Grant HD-10595.     

The inheritance of tissue-specific lactate dehydrogenase isozymes in interspecific bass (Micropterus) hybrids

A combination of hybridization (in vivo and in vitro), immunochemical, and electrophoretic analyses reveals that both smallmouth bass, Micropterus dolomieui (Lacépède), and largemouth bass, M. salmoides (Lacépède), possess three homopolymeric lactate dehydrogenase (LDH) isozymes, A₄, B₄, and E₄. The retinal-specific E₄ isozymes of these two parental species possess different electrophoretic mobilities. The two bass species were hybridized to produce the interspecific F₁ hybrids. In addition, F₂ and F₃ hybrid generations were produced. The genetic data from these crosses indicate that the retinal-specific LDH isozyme is the product of a distinct nuclear gene (E locus) on an autosomal chromosome. This E gene appears to segregate independently of the gene for supernatant MDH. The LDH E gene is highly active in the bass neural retina and less active in other neural tissues. However, unlike in most teleosts, the bass LDH E gene also functions in such nonneural tissues as the heart and kidney.This research was supported by NSF grant GB 16425 to G. S. Whitt and by funds provided by the Illinois Natural History Survey to W. F. Childers.     

Genetic analysis of cinnamyl alcohol dehydrogenase in loblolly pine: single gene inheritance, molecular characterization and evolution

The gene encoding the monolignol biosynthetic enzyme cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195) can be expressed in response to different developmental and environmental cues. Control of Cad gene expression could involve either differential regulation of more than one Cad gene or, alternatively combinatorial regulation of a single Cad gene. In loblolly pine (Pinus taeda L.), we found several electrophoretic variants (allozymes) of CAD and a high level of heterozygosity (h_e=0.46). Analysis of inheritance patterns of pine CAD allozymes gave segregation ratios that were consistent with Mendelian expectations for a single functional gene. The identity of the full-length Cad cDNA sequence was confirmed by alignment with peptide sequences obtained from purified active enzyme and by extensive similarity to Cad sequences from other species. Southern blot analysis of genomic DNA using the Cad cDNA as a hybridization probe gave simple patterns, consistent with our interpretation that pine Cad is a single-copy gene. Phylogenetic analysis and evolution rate estimates showed that Cad sequences are diverging less rapidly in the gymnosperms than in the angiosperms. The Cad mRNA was present in both lignifying tissues and a non lignifying tissue (the megagametophyte) of pine. The presence of a single gene suggests that different regulatory mechanisms for a single Cad gene, rather than differential regulation of several genes, can account for its expression in response to different cues.     

Patterns of gene expression during teleost embryogenesis: Lactate dehydrogenase isozyme ontogeny in the medaka (Oryzias iatipes)

The ontogeny of the lactate dehydrogenase (LDH; EC 1.1.1.27) isozymes during medaka (Oryzias latipes) embryogenesis was determined after the genetic and molecular bases of this multilocus isozyme system were established. Three LDH loci are differentially expressed among the tissues of the adult medaka. The LDH-A locus was expressed almost exclusively in the white skeletal muscle, the LDH-B locus in all tissues examined, and the LDH-C locus in the eye and brain. The contribution of each of these LDH loci was quantitatively determined throughout early medaka embryogenesis by using a combination of electrophoretic, immunochemical, and spectrophotometric procedures. LDH-B₄ is present throughout embryogenesis and is the predominant LDH isozyme during this period. LDH-C subunit activity was first detected 146 hr after fertilization (26°C), 142 hr prior to hatching. LDH-A subunit activity, however, was not detected until after hatching and, then, only as heterotetramers containing LDH-B subunits. The pattern of LDH gene expression during medaka embryogenesis was compared with the patterns of LDH gene expression during early development in five other teleost species. Some common patterns of differential LDH gene expression appear to exist among the teleosts. In all species examined, isozymes encoded in at least one LDH locus, A and/or B, were present throughout development. Those isozymes present continually during embryogenesis also tend to be active in a wide variety of differentiated tissues in the adult fish. Conversely, LDH isozymes which are active in a restricted number of adult tissues are detected only later in embryogenesis. The initiation of LDH-C gene expression, however, is closely coupled with morphological and functional differentiation of those cells in which this locus is predominantly expressed in the adult.     

LDH isozymes in amazon fish—III. Distribution patterns and functional properties in Serrasalmidae (Teleostei: Ostariophysi)

• 1.1. The patterns of distribution of LDH isozymes in five tissues from 12 Serrasalmidae species have been studied through two-fold serial dilution (Klebe's test) as well as the pyruvate inhibition of LDH enzyme in skeletal and heart muscles (low/high ratios).
• 2.2. The species' electrophoretic patterns differ by orthologous A₄ isozyme mobilities since the orthologous B₄ isozymes present similar electrophoretic migration. These differences between Ldh-A and Ldh-B products reflect three-, four-, and five-banded patterns. Thus, different LDH isozyme numbers formed from A and B subunits should not be used as an evolutionary or phylogenetic characteristic from different taxonomic groups.
• 3.3. Out of 66 pairs of species only five pairs showed significant differences in the distribution patterns in all five analyzed tissues, while no pair of species showed the same distribution in these tissues. This variation was explained as differential regulation of structural genes among tissues and/or species.
• 4.4. Functional properties showed significant between the LDHs from heart and skeletal muscles, and are consistent with a preference for aerobic metabolism. We suppose that by selecting B-like subunits these fishes are able to maintain good control of aerobic/anaerobic transitions, maintaining predominantly oxidative metabolism even in hypoxic waters, with which they have to cope.

     

New members of the neurexin superfamily: multiple rodent homologues of the human CASPR5 gene

Proteins of the Caspr family are involved in cell contacts and communication in the nervous system. We identified and, by in silico reconstruction, compiled three orthologues of the human CASPR5 gene from the mouse genome, four from the rat genome, and one each from the chimpanzee, dog, opossum, and chicken genomes. Obviously, Caspr5 gene duplications have taken place during evolution of the rodent lineage. In the rat, the four paralogues are located in one chromosome arm, Chr 13p. In the mouse, however, the three Caspr5 genes are located in two chromosomes, Chr 1 and Chr 17. RT-PCR shows that all three mouse paralogues are being expressed. Common expression is found in brain tissue but different expression patterns are seen in other organs during fetal development and in the adult stage. Tissue specificity of expression has diverged during evolution of this young rodent gene family.     

Evolution of patterns of gene expression in Hawaiian picture-wingedDrosophila

Summary The tissue and stage specificity of expression of five enzymes was examined by electrophoretic analysis of relative enzyme levels in extracts of 13 larval and adult tissues in 27 species of Hawaiian picture-wingedDrosophila. The developmentally regulated patterns of enzyme expression thus characterized were compared to a modal standard phenotype. About 30% of the pattern features analyzed differed significantly from the standard in one or more species. Many of these regulatory differences are essentially qualitative, with tissue specific differences in enzyme activity in excess of 100 fold for some species pairs. The adaptive significance of these pattern differences is unknown, but the results provide strong direct evidence for rapid evolution of new patterns of gene regulation in this group of organisms.     

Phagocytic Activity of Three Naegleria Strains in the Presence of Erythrocytes of Various Types1

The phagocytic activities of N. lovaniensis (Aq/9/1/45D) and N. gruberi (1518/1f and 1518/1e) were studied in the presence of erythrocytes of various species: chicken, rabbit, goat, and human (A⁺, B⁺, and AB⁺ were tested). The percentage of amoebae with ingested red cells, the phagocytic index (PhI), can be considered as an expression of phagocytic activity. Under given conditions (erythrocyte concentration, incubation time, age of amoebic cultures) each strain of Naegleria prefers one erythrocyte type. Thus, for 72-h cultures, N. lovaniensis ingested more A⁺ type erythrocytes than did N. gruberi strains but had very low affinity for rabbit red cells except when very high concentrations were tested. Naegleria gruberi 1f was the most active of the three strains towards rabbit and B⁺ and AB⁺ human erythrocytes, but very low PhIs were obtained with goat erythrocytes. Naegleria gruberi le exhibited high phagocytic activity for every erythrocyte type except for rabbit red cells.     

Role of the B-G-region antigen in the humoral immune response to the B-F-region antigen of chicken MHC

In the chicken MHC there exist two regions, designated F and G, which were separated by crossing-over. The F region contains genes controlling all functions characteristic of the MHC. So far only one gene has been assigned to the G region and it is responsible for the presence of an RBC antigen. When cross immunizing animals of the congenic lines CB and CC with erythrocytes, we have found that both F- and G-specific antibodies were produced. By using the recombinant haplotypes B ^R1 and B ^R2 we were able to dissociate the F from the G antigen and immunize with them separately. It was found that production of F antibodies required the copresence of the G antigen, whereas G antibodies were formed regardless of the presence or absence of the F-region antigen. It could be demonstrated that a prerequisite of the role of the G antigen with respect to the F antigen was the localization of both antigens on the same erythrocyte. Possible mechanisms underlying this phenomenon are discussed.Abbreviations used in this paper MHC major histocompatibility complex - RBC red blood cells - PBL peripheral blood lymphocytes - GVH graft-versus-host - MLR mixed lymphocyte reaction - i.v. intravenous - PBS phosphate-buffered saline - ME mercaptoethanol     

Brain and erythrocyte microtubules from chicken contain different beta-tubulin polypeptides

beta-Tubulin subunits isolated from chicken brain tissue and erythrocytes are distinguishable as unique biochemical species by electrophoretic and peptide mapping procedures. 1) The subunits of beta-tubulin exhibit major differences in electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels that vary according to the pH and ionic strength of the gel. 2) The isoelectric points of urea-denatured beta subunits from brain tissue and erythrocytes are pH 5.1 and 5.4, respectively, whereas those of both alpha subunits are approximately pH 5.2.3) Two-dimensional peptide maps prepared with alpha-chymotrypsin or V8 protease show that alpha-tubulin peptides are indistinguishable, whereas beta-tubulin peptides are very different. Only one-third of the 15 major tyrosine-containing beta-tubulin peptides prepared with alpha-chymotrypsin are common to both beta-tubulin species. The data indicate that the beta-tubulin subunits of brain tissue and erythrocytes are biochemically distinct and may be different gene products. The presence of tubulin variants in brain tissue and erythrocytes may indicate special requirements for microtubule assembly and function in different cell types.     

Evolutionary changes in sites and timing of actin gene expression in embryos of the direct- and indirect-developing sea urchins, Heliocidaris erythrogramma and H. tuberculata

 We describe an evolutionary comparison of expression of the actin gene families of two congeneric sea urchins. Heliocidaris tuberculata develops indirectly via a planktonic feeding pluteus that forms a juvenile rudiment after a long period of larval development. H. erythrogramma is a direct developer that initiates formation of a juvenile rudiment immediately following gastrulation. The developmental expression of each actin isoform of both species was determined by in situ hybridization. The observed expression patterns are compared with known expression patterns in a related indirect-developing sea urchin, Strongylocentrotus purpuratus. Comparisons reveal unexpected patterns of conserved and divergent expression. Cytoplasmic actin, CyIII, is expressed in the aboral ectoderm cells of the indirect developers, but is an unexpressed pseudogene in H. erythrogramma, which lacks aboral ectoderm. This change is correlated with developmental mode. Two CyII actins are expressed in S. purpuratus, and one in H. erythrogramma, but no CyII is expressed in H. tuberculata despite its great developmental similarity to S. purpuratus. CyI expression differs slightly between Heliocidaris and Strongylocentrotus with more ectodermal expression in Heliocidaris. Evolutionary changes in actin gene expression reflect both evolution of developmental mode as well as a surprising flexibility in gene expression within a developmental mode. Received: 27 July 1997 / Accepted: 30 December 1997     

Differential expression for tissue-specific isozymes in three species of Hypostomus Lacépède, 1803 (Teleostei: Loricariidae)

The expression patterns of 14 enzymatic systems in skeletal muscle, liver and heart tissues of three species of Hypostomus from the Iguaçu River basin (Brazil) were investigated. Although the patterns were similar for the three species, different tissues showed differential expressions, and the data showed that differential tissue expressions of isoperoxidases may be due to preferential combination or association of polypeptide subunits. The detected patterns for SOD isozymes showed that the quaternary structures of these enzymes were in disagreement with the subunit number reported for the majority of other vertebrate groups. Tissue-specific restriction on heterotetramer formation also were described in LDH and MDHP isozymes. Thus, these tissue-specific gene expression character in the species of Hypostomus have the greatest potential to be recognized and applied in systematic studies among species of Hypostomus.     

A genetically controlled lactate dehydrogenase variant at the B locus in mink

Erythrocytes of 119 mink, and tissue extracts of three mink, were examined for electrophoretic patterns of lactate dehydrogenase (LDH). A variant was detected at the B locus. There are two alleles, LDH-B ^a and LDH-B ^b; three phenotypes, LDH-Ba, LDH-Bab, and LDH-Bb; and three genotypes, LDH-B ^a/LDH-B^a, LDH-B^a/LDH-B^b, and LDH-B ^b/LDH-B^b. The inheritance as observed in 24 families agrees with an autosomal, codominant, two-allele system at the LDH B locus.Supported by National Research Council Grant A-4442 and the Ontario Department of Agriculture and Food.     

Genetic control of LDH isozymes in the house fly,Musca domestica

Electrophoretic variations in lactate dehydrogenase from adult whole body homogenates are described for three laboratory strains of house fly, Musca domestica. Several crosses between different electrophoretic forms provided evidence that the observed variations are due to segregation of alleles at two distinct loci (designated as A and B loci) and that the LDH isozymes of house flies are dimers formed by a random association of subunits controlled by the two loci.     

Origin of human triosephosphate isomerase isozymes: Further evidence for the single structural locus hypothesis with Japanese variants

Summary Four electrophoretic variants of human erythrocyte triosephosphate isomerase (TPI) have been studied to investigate the origin of the multiple forms of human TPI, in particular the constitutive TPI-B isozyme and the cell division-associated TPI-A isozyme. The variant phenotype expressed by the constitutive TPI-B isozyme in both erythrocytes and peripheral lymphocytes was also expressed by the cell division-associated isozymes in mitogen-stimulated lymphocytes and hair root cells. These results strongly support the hypothesis of Decker and Mohrenweiser (1981) that TPI-B and TPI-A originated from the same structural gene. We also found that the isozyme e is different from TPI-A with respect to both its electrophoretic mobility and heat stability. This finding is in contrast to the recent conclusion of Yuan et al. (1981) that both the isozyme e and TPI-A are deamidation products of TPI-B.     

Recurrent decreases of erythrocyte electrophoretic mobility in rats exposed to 1.0 R whole-body X-radiation

Circulating erythrocytes from rats were examined up to 30 weeks post whole-body exposures of 1.0 R for alterations in the expression of net negative surface charge as measured by whole-cell microelectrophoresis in saline sorbitol. Erythrocyte electrophoretic mobility was determined in an apparatus composed of a horizontal transilluminated cylindrical chamber, equipped with a reversible, blacked platinum electrode, immersed in a water bath maintained at 25.0±0.1°C (Rank Brothers). In two separate experiments, recurrent decreases in the expression of net negative surface charge occurred at 10, 17, and 30 weeks post-irradiation. At these times distribution analyses of recorded erythrocyte electrophoretic mobility (EEPM) values revealed a skewing of the normally distributed EEPM population values to lower EEPM. Total sialic acid content released from hydrolyzed erythrocyte membrane preparations revealed no significant differences between erythrocytes from sham and irradiated animals. In vivo post-irradiation labeling of erythrocytes with diisopropyl-[³²P] phosphorofluoridate at 4 and 33 weeks (separate experiments) indicated only a minor abbreviated erythrocyte life span at 33 weeks. Therefore, effects from low dose (1.0 R) whole-body irradiation would appear to include a recurrent defect in the expression of the net negative surface charge.     

Characterisation of two gene subunits on the 1R chromosome of rye as orthologs of each of the Glu-1 genes of hexaploid wheat

The visco-elastic properties of bread flour are firmly associated with the presence or absence of certain HMW subunits coded by the Glu-1 genes. Identifying allelic specific molecular markers (AS-PCR) associated with the presence of Glu-1 genes can serve as a valuable tool for the selection of useful genotypes. This paper reports the use of primers designed from nucleotide sequences of the Glu-D1 gene of wheat (AS-PCR for Glu-D1y10) that recognise and amplify homologous sequences of the Glu-R1 gene subunits of rye. The primers amplify the complete coding regions and provided two products of different size in rye, in wheats carrying the substitution 1R(1D) and in rye-wheat aneuploid lines carrying the long arm of chromosome 1R. The location, the molecular characterisation of these sequences and their expression during grain ripening seem to demonstrate that the amplification products correspond to structural genes encoding the high-molecular-weight (HMW) glutenins of rye. The homology of the rye gene to subunits encoding HMW glutenins in wheat was confirmed by Southern blots and sequencing. The amplification-products were cloned, sequenced and characterised, and the sequences compared with the main glutenin subunits of wheat and related species. Further, an RT-PCR experiment was performed using primers designed from the sequence of both amplified products. This assay demonstrated that both sequences are expressed in endosperm during grain ripening. The results of these analyses suggest that both gene subunits correspond to x- and y-type genes of the Glu-R1 locus of rye. Received: 11 December 2000 / Accepted: 17 April 2001     

Lactate dehydrogenase gene function in the blind cave fish,Anoptichthys jordani,and other characins

The functions of the genes encoded for lactate dehydrogenase (LDH) in six genera of characins (teleost) were examined by electrophoretic and immunochemical analyses of the LDH isozymes. The characins possess the LDH A and B loci present in all vertebrates. The eyeless Mexican cave fish (Anoptichthys jordani) and other characins possessing normal eyes, e.g., Mexican tetra (Astyanax mexicanus, which is able to hybridize with the cave fish), blue tetra (Mimagoniates microlepis), sailfin tetra (Crenuchus spilurus), head and tail light tetra (Hemigrammus ocellifer), and the piranha (Serrasalmus spilopleura), all lack the function of a third LDH locus (the E locus) present in many teleosts which codes for a distinctive isozyme synthesized in the nervous system, particularly in the neural retina.This research was supported by NSF grant GB 16425 to G.S.W. F.S.M. was a James Scholar at the University of Illinois during the course of this research.     

Expression of a C3 plant Rubisco SSU gene in regenerated C4 Flaveria plants

We report the successful transformation, via Agrobacterium tumefaciens infection, and regeneration of two species of the genus Flaveria: F. brownii and F. palmeri. We document the expression of a C₃ plant gene, an abundantly expressed ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit gene isolated from petunia, in these C₄ plants. The organ-specific expression of this petunia gene in Flaveria brownii is qualitatively identical to its endogenous pattern of expression.