Abstracts

A new allele of peptidase-B in cattle

Maria Aparecida Cassiano Lara ¹ and Eucleia Primo B. Contel ²

¹Seção de Reprodução e Inseminação Artificial, Instituto de Zootecnia, Caixa Postal 60,

13141-000 Nova Odessa, SP, Brasil.

²Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, USP,

14049-900 Ribeirão Preto, SP, Brasil. Send correspondence to E.P.B.C.

ABSTRACT

Electrophoretic analyses of peptidase-B were carried out on red cell hemolysates from Holstein, Mantiqueira and Gyr cattle, using cornstarch, known in Brazil as Penetrose-30. We describe a new peptidase-B allele, denoted Pep-B1, in Mantiqueira cattle, belonging to the Bos taurus group, which are the result of a cross of native cattle of Portuguese origin introduced in Brazil during colonial times (16th century) with Holstein and Caracu cattle. The genetic control of peptidase-B was determined by typing parents and progeny segregating for all three alleles, confirming that peptidase B is controlled by a single autosomal locus with three codominant alleles, denoted Pep-B1, Pep-B2 and Pep-B3 The use of the citrate-phosphate buffer system, at pH 5.9, on 14% gel, under the electrophoretic conditions standardized in this study permitted good visualization of all peptidase-B variants.

INTRODUCTION

Protein polymorphisms, which represent part of the genetic variability existing among living beings, have been extensively used in studies of racial characterization (Tagliaro et al., 1995), of phylogenetic relations (Zafindrajaona and Lauvergne, 1993), and in studies of the linkage between these genetic markers and genes that affect traits of economic interest (Clamp et al., 1992).

In cattle, some polymorphic gene systems may be considered racial markers since certain alleles exclusively occur in breeds of Indian origin (Bos indicus). The AlbC allele of albumin (Ashton and Lampkin, 1965), the CaZ allele of carbonic anhydrase (Penedo et al., 1982) and the Pep-B1 allele of peptidase-B (Del Lama et al., 1992) are particularly important among them. Other systems in addition to these are considered to be excellent genetic markers because of the occurrence of certain alleles at distinct gene frequencies that permit the racial characterization of Bos taurus and Bos indicus groups. Among them are the Am-lC allele of amylase (Ashton, 1965; Gebicke-Härter and Geldermann, 1977), the TfE and TfD alleles of transferrin (Baker and Manwell, 1980; Jain et al., 1992), the GcC allele of the vitamin D-bound protein and Ptf-2S allele of posttransferrin-2 (Van de Weghe et al., 1982), and the NPH allele of nucleoside phosphorylase (Panepucci et al., 1990). Other studies have also demonstrated associations between these genetic markers and traits of economic interest, such as milk yield (Bovenhuis et al., 1992), beef production (Rahman and Kalan, 1986) and adaptive traits (Carr et al., 1974; Panepucci et al., 1989). These data demonstrate the importance of looking for new genetic markers that might permit a better understanding of the origin of cattle breeds, as well as their application to animal breeding.

MATERIAL AND METHODS

A total of 472 Holstein, Mantiqueira and Gyr dairy cows were sampled at random from herds belonging to the Instituto de Zootecnia de Nova Odessa and to the Estação Experimental of Pindamonhangaba and Ribeirão Preto, all of them in the State of São Paulo.

Blood samples were collected into 5-ml vacutainer tubes containing sodium EDTA as an anticoagulant. Red cells were separated by centrifugation at 2,000 g for 10 min, washed three times in 0.85% NaCl and stored at -20oC until the time for electrophoretic analysis.

Red cells were then incubated with an identical volume of 50 mM 2-mercaptoethanol for 20 min and the hemolysates were absorbed with 0.5 x 0.6 cm Whatman filter paper and applied to the gel.

The buffer system used in the cuvettes was phosphate/citrate, pH 5.9 (0.245 M NaH2PO4 and 0.15 M citric acid). The 14% gel (Val et al., 1981) was prepared by 1:40 dilution. Electrophoresis was run for approximately 15 h at 8oC, with 5 V/cm applied to the gel. The gels were then cut horizontally and developed with l-leucylglycyl-glycine as substrate (Lewis and Harris, 1967).

When a new band was detected, especially in Mantiqueira cattle, the progeny of all individuals that carried that phenotype was typed in order to exclude the possibility that the event observed was a secondary band. The progeny of mating between parents carrying the three alleles was also analyzed.

Gene frequencies and their respective standard deviations and Hardy-Weinberg equilibrium were determined using the Fregen computer program of the Genioc system (Cabello and Krieger, 1991).

RESULTS

The new method permitted the detection of a new peptidase-B allele, denoted Pep-B3, in Mantiqueira cows. The electrophoretic pattern of this enzyme, visualized with the substrate l-leucylglycyl-glycine, is illustrated in Figure 1.

Figure 1 - Electrophoretic pattern of cattle red cell peptidase-B obtained on 14% cornstarch gel, citrate/phosphate buffer (0.245 M NaH₂PO₄ and 0.15 M citric acid), pH 5.9, and developed with l-leucylglycyl-glycine as substrate. The data are presented from left to right. Pep-B 1-1 (lane 1- provenance Gyr); Pep-B 2-2 (lane 3); Pep-B 3-3 (lane 4); Pep-B 1-2 (lane 6); Pep-B 1-3 (lanes 2 and 7) and Pep-B 2-3 (lane 5). The segregation of the three alleles in a family of the Mantiqueira cattle can be observed in the pedigree shown above.

Analysis of parents and progeny confirmed that peptidase-B polymorphism is determined by a single locus with three alleles: Pep-B1, Pep-B2 and Pep-B3. Their products present differences in electrophoretic mobility, with Pep-B3 being the most anodal enzyme and Pep-B2 the intermediate one.

Estimated gene frequency, standard deviations and c2 values, which permitted us to determine Hardy-Weinberg equilibrium, are presented in

DISCUSSION

We identified a new allele of red cell peptidase-B, denoted Pep-B3, in Mantiqueira cattle, as well as alleles Pep-B1 and Pep-B2. The method described here permitted good visualization of all variants of this enzyme. The choice of the type of support used was due to the fact that cornstarch, known in Brazil as Penetrose- 30, is locally manufactured and can be easily acquired at low cost. The buffer system and electrophoretic conditions used permitted good separation, as well as the identification of all phenotypes of red cell peptidase-B.

On the basis of the review published by Harris and Hopkinson (1976), who pointed out that the different peptidases (EC 3.4.11 or 13* and 3.4.13.9) can be identified by their different electrophoretic properties and affinities for specific substrates, and considering that the peptidase activity studied here was developed with the same substrate (l-leucylglycyl-glycine) as previously used by Lewis and Harris (1967) for human hemolysates, and by Saison (1973) and Del Lama et al. (1992) for cattle, we conclude that the enzyme studied here corresponds to that described by these investigators, who called it Pep-B.

In addition, care was taken to incubate the hemolysates with 2-mercaptoethanol prior to use, in order to avoid oxidation of sulfhydryl radicals present in the molecule of the enzyme by oxidized glutathione, a substance found in large amounts in red cells. This minimized formation of secondary bands.

We detected peptidase-B monomorphism in Holstein cattle, in agreement with data reported by Saison (1973), who described the absence of genetic variation in Charolais, Holstein, Simmenthal and Limosine cattle, as later confirmed by Del Lama et al. (1992), who suggested the fixation of Pep-B2 in these taurine breeds.

The genetic variability detected in Gyr cows agrees with the data reported by Del Lama et al. (1992), who first described genetic polymorphism of red cell peptidase-B. These investigators reported the occurrence of the Pep-B1 allele in Zebu cattle and suggested its use as a genetic marker for this racial group. However, our results demonstrate Pep-B1 in Mantiqueira cows, which belong to the Bos taurus group.

Red cell peptidase-B from Mantiqueira cows was studied for the first time in the present investigation. The origin of this cattle is somewhat obscure. According to Guaragna et al. (1984), the Mantiqueira breed was formed from crosses between Holstein sires and Caracu cows, a breed introduced on the occasion of the discovery of Brazil. Thus, we raise the hypothesis that the Pep-B3 allele is a marker of the Portuguese breed.

A possible explanation for the occurrence of the Pep-B3 allele in Mantiqueira cattle is that these cows are being selected for adaptive traits which are often contrary to the objectives of selection for yield traits in specialized breeds, a fact that would lead to the elimination of genetic variability at the peptidase locus, as detected in the present study for Holsteins and in other studies in the literature (Saison, 1973).

ACKNOWLEDGMENTS

The authors wish to thank the directors of the Estação Experimental de Zootecnia, Dr. José Ramos Nogueira and Dr. Luciano Ricardo Marcondes da Silva, and their staff for the assistance provided during blood collection, and Drs. Roberto Hauck Reichter and Lewis Joel Greene for valuable suggestions. We are particularly grateful to the scientific investigator Guilherme Paes Guaragna, in charge of the project "Breeding of Mantiqueira Type Dairy Cattle" for encouraging us to study these animals.

We are also indebted to Refinaria de Milho Brasil for their kind gift of the starch used in the present study.

This research was supported by CAPES, CNPq and by Instituto de Zootecnia.

Publication supported by FAPESP.

RESUMO

A peptidase-B eritrocitária foi estudada eletroforeticamente em bovinos das raças Holandesa, Mantiqueira e Gir, utilizando-se o amido de milho conhecido no Brasil como Penetrose-30. Detectamos um novo alelo desta enzima, denominado Pep-B3, no bovino Mantiqueira. Este gado é descendente de raças que foram introduzidas no Brasil no período da colonização (século XVI) e do gado Caracu e Holandês.

Determinamos o controle genético da peptidase-B através da análise da progênie de pais que possuíam os três alelos, confirmando que esta enzima é controlada por um único loco autossômico com três alelos,denominados Pep-B1, Pep-B2 e Pep-B3. A utilização de um tampão citrato-fosfato, pH 5,9 com o gel na concentração de 14% e nas condições eletroforéticas padronizadas neste trabalho, permitiu uma boa visualização de todas as formas da peptidase-B.

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(Received April 25, 1996)

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