Search

Whey Acidic Protein (WAP) has been identified in the milk of only a few species, including mouse, rat, rabbit, camel, pig, tammar wallaby, brushtail possum, echidna and platypus. Despite intensive studies, it has not yet been found in the milk of Ruminants. We have isolated and characterized genomic WAP clones from ewe, goat and cow, identified their chromosomal localization and examined the expression of the endogenous WAP sequence in the mammary glands of all three species. The WAP sequences were localized on chromosome 4 (4q26) as expected from comparative mapping data. The three ruminant WAP sequences reveal the same deletion of a nucleotide at the end of the first exon when compared with the pig sequence. Due to this frameshift mutation, the putative proteins encoded by these sequences do not harbor the features of a usual WAP protein with two four-disulfide core domains. Moreover, RT-PCR experiments have shown that these sequences are not transcribed and are, thus, pseudogenes. This loss of functionality of the gene in Ruminants raises the question of the biological role of the WAP. Some putative roles previously suggested for WAP are discussed.

The cysteine-rich secretory protein (CRISP) family consists of three members called acidic epididymal glycoprotein 1 (AEG1), AEG2, and testis-specific protein 1 (TPX1), which share 16 conserved cysteine residues at their C-termini. The CRISP proteins are primarily expressed in different sections of the male genital tract and are thought to mediate cell-cell interactions of male germ cells with other cells during sperm maturation or during fertilization. Therefore, their genes are of interest as candidate genes for inherited male fertility dysfunctions and as putative quantitative trait loci for male fertility traits. In this report, the cloning and DNA sequence of 137 kb of horse genomic DNA from equine chromosome 20q22 containing the closely linked equine TPX1 and AEG2 genes are described. The equine TPX1 gene consists of ten exons spanning 18 kb while the AEG2 gene consists of eight exons that are spread over 24 kb. The expression of these two genes was investigated in several tissues by reverse transcription polymerase chain reaction analysis and Western blotting. Comparative genome analysis between horse, human, and mouse indicates that all three CRISP genes are clustered on one chromosomal location, which shows conserved synteny between these species.

Acidic epididymal glycoprotein 1 (AEG1), also called cysteine-rich secretory protein 1 (CRISP1), is a member of the CRISP protein family which is characterized by 16 conserved cysteine residues at the C-terminus. The CRISP proteins are expressed in the male genital tract and are thought to be involved in sperm-egg fusion. Therefore, their genes are of interest as candidate genes for inherited male fertility dysfunctions and as putative quantitative trait loci for male fertility traits. In this report, the cloning and DNA sequence of 90 kb of horse genomic DNA from equine chromosome 20q22 containing the complete equine AEG1 gene are described. The equine AEG1 gene consists of eight exons spanning 31 kb. Analysis of equine AEG1 transcripts did not reveal any evidence for alternative splicing, however three different transcription start sites are used. The first transcription start site is located 20 nt downstream of a TATA box motif. Reverse transcription polymerase chain reaction analysis demonstrated that AEG1 is expressed in different parts of the epididymis, whereas it is hardly detectable in the testis. The naturally occurring diversity of the equine AEG1 gene in different horse breeds was investigated and several polymorphisms are reported, including one that affects the amino acid sequence. Finally, sequence comparisons revealed that the intronless equine PGK2 gene for the testis-specific phosphoglycerate kinase is located approximately 39 kb downstream of AEG1.

Several casein (CSN) genes (CSN1, 2, 10 and alphas2-CSN) have been described and shown to be clustered in mouse, man and cattle. These genes are expressed simultaneously in the mammary gland during lactation, but they are silent in most mammary cell lines, even in the presence of lactogenic hormones. However, it has been shown that the CSN2 gene, and this gene only, can be induced in certain mammary cell lines, such as HC11. In the present paper, we describe three overlapping bacterial artificial chromosome (BAC) clones which harbor both the rabbit CSN1 and CSN2 genes. These two genes are in a convergent orientation, separated by an intergenic region of 15 kb. DNA from one of the CSN/BAC clones was used as a probe for in situ hybridization to show that the CSN1 and CSN2 gene cluster is located on chromosome 15 band q23 and not on chromosome 12 as had been previously reported. Each of the three CSN/BAC DNAs was transfected into HC11 cells. In the presence of lactogenic hormones, the rabbit CSN1 gene was clearly expressed from all three CSN/BAC DNAs, whereas the rabbit CSN2 gene, which at the most possesses a 1 kb upstream region in one of the CSN/BAC DNAs, was not expressed at detectable levels on Northern blots. The transfected HC11 cells now express both rabbit CSN1 and mouse CSN2 genes. These transfected cells will be used as a model to study the role of CSN1 in milk protein secretion.