Abstract
Bovine antibodies have recently attracted increasing attention, as they have been shown to exhibit prophylactic and therapeutic properties in selected infectious diseases in humans. In the present study, we have isolated bacterial artificial chromosomes and cosmid clones containing the bovine JH, mu, delta, gamma 1, gamma 2, gamma 3, epsilon, and alpha genes, which allowed us to make a contig of the genes within the bovine IGHC locus. The genes are arranged in a 5'-JH-7 kb-mu-5 kb-delta-33 kb-gamma 3-20 kb-gamma 1-34 kb-gamma 2-20 kb-epsilon- 13 kb-alpha-3' order, spanning approximately 150 kb DNA. Examination of the bovine germline JH locus revealed six JH segments, two of which, JH1 and JH2, were shown to be functional although there was a strong preference for expression of the former. Sequence alignment of the bovine 5' E mu enhancer core region with those of other mammals, demonstrated an absence of the mu E3 motif and a shortened spacer between the mu A and mu B sites within the bovine E mu enhancer core region. Furthermore, the essential sequence element for class switching, switch mu, spanning approximately 3-kb repetitive sequence and abundant in the switch region motifs CTGGG (187 repeats) and CTGAG (127 repeats), was identified immediately upstream of the mu gene. A further sequence comparison revealed that the bovine IGHC genes display an extensive polymorphism leading to expression of multiple antibody allotypes.
Highlights
Bovine antibodies have recently attracted increasing attention, as they have been shown to exhibit prophylactic and therapeutic properties in selected infectious diseases in humans
Four BAC clones were obtained by screening of a BAC library [28], where BAC66R4C11 contained the , ␦, ␥3, ␥1 genes, BAC416R4C8 contained the , ␦, and a part of the C␥3 gene, BAC389R7C7 contained the ␥1 and ␥2 genes, while BAC412R7C5 contained only the ␥1 gene according to results based on Southern blotting and PCR analysis
The Bovine immunoglobulin heavy chain constant region gene (IGHC) Gene Locus Is Arranged in an Order of 5Ј-–␦–␥3–␥1–␥2–⑀–␣-3Ј—Since it was already known that the and ␦ genes are situated most 5Ј in the bovine IGHC gene locus [7, 14], the co-existence of the , ␦, and ␥3 genes in some BAC and cosmid clones suggested that the ␥3 gene may be the first constant region gene located downstream of the ␦ gene
Summary
Bovine BAC and Cosmid Libraries—Bovine IGHC gene-positive BAC clones were isolated from a previously constructed BAC library [28], which was generated using pBeloBAC11, a BAC vector. While the common forward primer bIgVH-leaders (P1, 5Ј-GCT CCA AGA TGA ACC CAC TGT G-3Ј) is derived from the leader exon of the VH genes, both bIgJH1as (P2, 5Ј-GAG GAG ACG GTG ACC AGG AGT-3Ј) and bIgJH2as (P3, 5Ј-GAG GAG ACG GTG ACC TCG ATC-3Ј) are JH gene-specific primers Both bovine genomic DNA (derived from blood, spleen, bone marrow) and cDNA (spleen) were employed as templates for PCR under conditions of 94 °C 3 min, 30 cycles of 94 °C 30 s, 62 °C 30 s, 72 °C 40 s, hold at 72 °C for 10 min. The PCR products were separated on an agarose gel and subsequently transferred to nylon membranes for hybridization with a bovine VH gene fragment generated by PCR from a cloned IgD heavy chain cDNA [14] using primers bIgVH-hys (5Ј-GTG CAG CTG CGG GAG TCA-3Ј) and bIgVH-hyas (5Ј-CTC AAC ATA AGG ACA AGC-3Ј). The dot plot comparison was performed using the same program
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