G. Cosenza

Molecular characterisation, genetic variability and detection of a functional polymorphism influencing the promoter activity of OXT gene in goat and sheep.

The purpose of the study described in this Research Communication was to report the full characterisation of the goat and sheep oxytocin-neurophysin I gene (OXT), their promoters and amino acid sequences. Using the genomic DNA as template, we sequenced and compared the whole OXT gene (3 exons), plus 958/960 nucleotides at the 5 flanking region and 478/477 nucleotides at the 3 flanking region, in 46 sheep and 24 goats belonging to different breeds/genetic types reared in Italy, Greece and Germany. The comparison of the obtained sequences showed a high degree of genetic variability at these loci. In particular, we focused on the SNP g.438T > C as possible example of trans-specific polymorphism. This SNP alters a putative binding site of the transcription factor Oct-1. The set-up of a luciferase assay confirmed that the C variant of this SNP negatively affects the promoter activity of the sheep OXT gene. The results of this study suggest that the SNP g.438T > C might be useful to promote association studies with traits/physiological processes controlled by this hormone.

Molecular cloning, promoter analysis and SNP identification of Italian Nicastrese and Saanen lactoferrin gene

Lactoferrin (Lf) is an iron-binding glycoprotein found in exocrine secretions including milk. High levels of lactoferrin may have a role in the prevention of microbial infection of the mammary gland. In this report we sequenced and characterized goat lactoferrin cDNA and its promoter region in two different breeds of goat. The complete cDNA comprised 2356 nucleotides, including 38 bp at the 5-UTR and 194 bp at the 3-UTR. The open reading frame is 2127 bp long and it encodes a mature protein of 689 aminoacids. A total of 19 nucleotide differences, 11 of them being responsible for 8 aminoacid changes, were identified through the comparison with French, Korean and Tibetan goat lactoferrin cDNAs. About 1700 bp of the lactoferrin gene promoter were sequenced. Sequence analysis revealed a non-canonical TATA box, multiple SP1/GC elements, and other putative binding sites for transcription factors, such as NF-kappaB, STAT3 and AP2. Two SNPs were identified, one of which would seem to create a new putative AP2 consensus sequence. The presence of an additional AP2 binding site could be associated with quantitative differences of such protein fraction, which could enhance all the activities related to such protein, and improve mammary gland defence against bacterial infections.

A preliminary analysis of the goat lactoferrin encoding gene

Riassunto Analisi preliminare del gene che codifica la lattoferrina caprina. È stato sequenziato il tratto di DNA comprendente gli ultimi 30 nucleotidi del 2° introne e i primi 52 nucleotidi del 6° introne (per un totale di 2824 bp) del gene della lattoferrina (Lf) di 3 capre appartenenti rispettivamente alla razza Saanen, Maltese e ad una popolazione autoctona allevata in provincia di Catanzaro (Italia). Il confronto tra le sequenze ottenute ha evidenziato 8 siti polimorfici (6 transversioni e 2 transizioni realizzatesi a livello intronico), mentre il confronto con le sequenze dei cDNA della Lf caprina depositate in Banca Dati mostra 5 sostituzioni nucleotidiche responsabili di tre sostituzioni aminoacidiche. In generale, il gene Lf caprino presenta una struttura simile a quella dell’omologo gene nella specie bovina, fatta eccezione per l’inserzione in quest’ultima specie di una sequenza di origine retroposonica (Bov A) a livello del 4° introne.

A preliminary analysis of mRNA transcribed from the N allele at the CSN1S1 locus of the goat

The goat αS1-casein is a main milk phosphoprotein of 199 aminoacid residues long. The αS1-casein gene (CSN1S1) contains 19 exons, ranging in size from 24 to 385 bp (for a total of 1138 bp) in the coding region, spread over about 17,5 kb transcription unit (Leroux et al., 1992). The goat αS1-casein exhibits both qualitative and quantitative remarkable variation arising from genetic polymorphism in the encoding gene. In particular, two alleles, CSN1S1 01 and 02, associated with a null content of the corrisponding protein, were detected (Martin et al., 1999), characterized by large DNA deletion (Cosenza et al., 2002) and insertion (Martin et al., 1999) events, respectively. Recently, by means of SDS-PAGE and CSN1S1 gene sequence analysis of goat individual milk and DNA sample, another allele (CSN1S1 N), associated with an apparently null amount of this protein fraction in the milk, was identified (Ramunno et al., 2002). This allele is characterized by the citosine deletion at position 23 in the 98th exon of the gene, likewise to CSN1S1 F allele (associated to a low content of αS1- casein) and, contrary to this one, for the absence of two insertions, 11 and 3 bp base pair in length, in the downstream intron. The present work reports the results of a preliminary analysis of mRNA transcribed from the N allele at goat CSN1S1 locus compared with the one transcribed from a homozygote individual for the CSN1S1 A allele.