Haplotype combinations of <Emphasis Type="Italic">AdPLA</Emphasis> gene polymorphisms associate with growth traits in Chinese cattle

Ablation of AdPLA in mice could prevent obesity, which implicated AdPLA was a new candidate gene for growth traits. In this study, we screened the polymorphisms of AdPLA gene and revealed three novel SNPs (NC_007330.4:g.43638506 C>T, 43658457 T>C and 43661404 T>C) by DNA sequencing and forced PCR-RFLP methods in 1340 individuals from six breeds. A preliminary single SNP-traits association analysis demonstrated that CC genotype was significantly higher than TT genotype at two growth traits in P6-EcoRII locus (P < 0.05). Moreover, the P8-FbaI locus had a significant association with some growth traits, individuals with TT genotype were higher than CC genotype (P < 0.05). A further analysis confirmed that these two SNPs were in linkage disequilibrium, and haplotype H2, both advantage allelic in P6-EcoRII and P8-FbaI loci, was better than the others for bovine growth traits, which was consistent with single SNP-traits association results. Therefore, this study not only proved that these SNPs were in association with bovine growth traits, but also contributed to evaluate them as genetic markers in bovine genetics and breeding and had potential application in breeding programs.     

Single nucleotide polymorphisms in bovine <Emphasis Type="Italic">PRL</Emphasis> gene and their associations with milk production traits in Chinese Holsteins

Prolactin (PRL) plays a crucial role in the initiation and maintenance of lactation in mammals. In this study, seven PCR fragments representing most important functional domains of PRL gene were screened for single nucleotide polymorphisms (SNPs) in Chinese Holsteins by single-strand conformation polymorphisms and amplicons sequencing, and their genetic effects on milk production traits were evaluated. A total of four SNPs, including two in the promoter (−1043A>G and −402A>G), one in intron 1 (+2723C>T) and one in exon 4 (+8398G>A) were identified in PRL gene. Statistical results showed significant associations between the promoter genotypes and the milk performance traits in Chinese Holsteins. Cows with genotype P1-GG showed higher milk yields (P < 0.01), while cows with genotype P1-AA showed higher fat contents (P < 0.01). Haplotype analysis of two SNPs in promoter region revealed that the Hap(AG) was significantly associated with increased milk yields and Hap(AA) was associated with increased fat contents (P < 0.01). This is the second study reporting SNPs in the 5′-regulatory region of PRL gene, which interfere with milk production traits.     

Effects of genetic variability of the dairy goat growth hormone releasing hormone receptor (<Emphasis Type="Italic">GHRHR</Emphasis>) gene on growth traits

Growth hormone-releasing hormone receptor (GHRHR) plays a critical role in growth hormone (GH) synthesis, release and regulation of pituitary somatotroph expansion in vertebrates. The objective of this study was to investigate variations in goat GHRHR gene and their associations with growth traits in 668 dairy goats. The results showed four novel single nucleotide polymorphisms (SNPs): NC_007302:g.5203C>T, 7307C>G, 9583G>A and 9668A>C. In detail, the novel SNP C>T in the 5203rd nucleotide identified a missense mutation: CCC (Pro)>TCC (Phe) at position 116aa of the goat GHRHR (423aa). Besides, 9583G>A and 9668A>C polymorphism were in complete linkage disequilibrium. The genetic diversity analysis revealed that the Guanzhong dairy goat possessed intermediate genetic diversity in P3 and P7 loci, and the Xinong Sannen dairy goat belonged to poor genetic diversity in P4 locus. Significant associations between the genotypes of P3 locus and body length, body height and chest circumference was observed in Guanzhong goat (P < 0.05). However, in Xinong saanen population, significant statistical difference was only found in body height and body length (P < 0.05). In P4 and P7 loci, no significant associations were detected between any variant sites and body length, body height and chest circumference, as well as for the milk traits (P > 0.05). These results strongly suggested that the goat GHRHR gene is a candidate gene that influences growth traits in dairy goat.     

Polymorphisms of <Emphasis Type="Italic">STAT5A</Emphasis> gene and their association with milk production traits in Holstein cows

The STAT5A gene was studied as a candidate gene for five milk production traits (milk yield at 305 days, protein percentage, fat percentage, lactose percentage and dry matter percentage) in Holstein cows. According to the sequence of bovine STAT5A gene, two pairs of primers (P1 and P2) were designed to detect polymorphisms of STAT5A gene in 401 Holstein cows by PCR-RFLP and PCR-SSCP. The results showed that the products amplified by primers P1 and P2 displayed polymorphisms. For P1, three genotypes (AA, AG, and GG) were detected, and the frequency of AA/AG/GG was 0.252/0.486/0.262, respectively. Sequence analysis revealed a single nucleotide substitution A–G at 14217 bp (GenBank NC_007317) of bovine STAT5A gene while compared GG genotype with AA genotype. The differences of the least squares means for the four milk production traits (milk yield at 305 days, fat percentage, lactose percentage and dry matter percentage) between AA, AG and GG were not significant (P > 0.05). Least squares mean of protein percentage for AG or GG was significantly higher than that for AA (P < 0.05); the difference of the least squares mean for protein percentage was not significant between AG and GG (P > 0.05). For P2, three genotypes (CC, CT, and TT) were detected in Holstein cows, and the frequency of CC/CT/TT was 0.751/0.234/0.015, respectively. Sequencing revealed an insertion CCT at 17266 (NC_007317) of bovine STAT5A gene while compared CC genotype with TT genotype. The differences of the least squares means for the three milk production traits (protein percentage, lactose percentage and dry matter percentage) between CC, CT and TT were not significant (P > 0.05). Least squares mean of milk yield at 305 days for TT or CT was significantly higher than that for CC (P < 0.05); the difference of the least squares mean for milk yield at 305 days was not significant between TT and CT (P > 0.05). Least squares mean of fat percentage for CC or CT was significantly higher than that for TT (P < 0.05); the difference of the least squares mean for fat percentage was not significant between CC and CT (P > 0.05). The results preliminarily indicated that allele G of A14217G polymorphic site of STAT5A gene is a potential DNA marker for improving protein percentage in dairy cattle, 17266indelCCT polymorphic site of STAT5A gene is a potential DNA marker for improving milk yield at 305 days and fat percentage in dairy cattle.     

Identification of three novel SNPs and association with carcass traits in porcine <Emphasis Type="Italic">TNNI1</Emphasis> and <Emphasis Type="Italic">TNNI2</Emphasis>

In this study, two novel SNPs (EU743939:g.5174T>C in intron 4 and EU743939:g.8350C>A in intron 7) in TNNI1 and one SNP (EU696779:g.1167C>T in intron 3) in TNNI2 were identified by PCR–RFLP (PCR restriction fragment length polymorphism) using XbaI, MspI and SmaI restriction enzyme, respectively. The allele frequencies of three novel SNPs were determined in the genetically diverse pig breeds including ten Chinese indigenous pigs and three Western commercial pig breeds. Association analysis of the SNPs with the carcass traits were conducted in a Large White × Meishan F₂ pig population. The linkage of two SNPs (g.5174T>C and g.8350C>A) in TNNI1 gene had significant effect on fat percentage. Besides these, the g.5174T>C polymorphism was also significantly associated with skin percentage (P < 0.05), shoulder fat thickness (P < 0.05) and backfat thickness between sixth and seventh ribs (P < 0.05). The significant effects of g.1167C>T polymorphism in TNNI2 gene on fat percentage (P < 0.01), lean meat percentage (P < 0.05), lion eye area (P < 0.05), thorax–waist backfat thickness (P < 0.01) and average backfat thickness (P < 0.05) were also found.     

Effects of genetic variability of the carpine homeobox transcription factor <Emphasis Type="Italic">HESX1</Emphasis> gene on performance traits

HESX1 plays a key role in the development of the forebrain and pituitary gland and produces potential effects on performance traits. The objective of this study was to detect and assess the associations of the possible polymorphisms of six loci within HESX1 gene with performance traits in Chinese 1,119 goats. Only one novel SNP (NM_001494116:g.307049A > G) locating on IVS1 + 348A > G was identified and detected by HaeIII forced-RFLP-PCR. The frequencies of allele “G” varied from 0.025 to 0.245 in analyzed populations with the Hardy–Weinberg equilibrium (P > 0.05). Genotypic and allelic frequencies were found to be significantly different in four breeds (χ² = 147.674, df = 6, P < 0.001; χ² = 157.250, df = 3, P < 0.001, respectively), implying that the distribution of genotypic and allelic frequencies of goat HESX1 gene was significantly associated with different goat utilities (cashmere, meat and dairy). Association analysis results revealed no significant effects of caprine HESX1 gene on body sizes in XNSN population (P > 0.05) and cashmere traits in IMWC population (P > 0.05). Significant statistical of HESX1 gene with body weight was found (*P < 0.05). The genotype AA showed significantly higher body weight than those of AG in 2-year-old age (*P < 0.05), while the AA genotype was senior to AG genotype in 4-year-old body weight trait (*P < 0.05). These suggestions indicated that the HESX1 gene has significant effect on goat body weight depending on ages, which is accordance with the function repressor of the HESX1.     

Two novel cSNPs of <Emphasis Type="Italic">weaver</Emphasis> gene in Chinese indigenous goat and their associations with milk yield

Weaver gene plays an essential physiological role in the function of many organs, including brain, heart, kidney and endocrine cells, and also in the regulation of insulin secretion by glucose and/or neurotransmitters. Thus, weaver gene is an important potential candidate gene effecting on performance traits. The objective of this study was to detect the genetic variation of five loci within weaver gene by PCR-SSCP, DNA sequencing and forced PCR-RFLP methods in 1,019 Chinese indigenous goats. Two novel coding SNPs (XM_598993:m.864G>A; XM_598993:m.1224T>A) locating on P3 and P4 loci were identified and detected by MluI and AsuII forced PCR-RFLP, respectively. In the MluI analysis, the frequencies of goat MluI-A allele in the analyzed populations were 0.226, 0.248, 0.096 and 0.088 for XNSN, GZ, SBWC and XJWC, respectively. Genotypic frequencies were found to be significantly different in four breeds (χ² = 75.842, df = 6, P < 0.001); In the AsuII analysis, the frequencies of goat AsuII-A allele in the analyzed populations were 0.584, 0.441, 0.073 and 0.063 for XNSN, GZ, SBWC and XJWC, respectively. Genotypic frequencies were found to be significantly different among four breeds (χ² = 399.464, df = 6, P < 0.001). The frequencies of allele MluI-A and AsuII-A in XNSN and GZ populations were significantly higher than those of SBWC and XJWC goats. Association analysis with adjusted milk yield in the XNSN breed indicated that the animals with AsuII-AA genotype owned significantly higher adjusted milk yield than the ones with AsuII-TT genotype in the second lactation (P < 0.05). The observation suggested that the allele “AsuII-A” had the positive effects on adjusted milk yield in the second lactation.     

Molecular characterization and polymorphisms of the caprine Somatostatin (<Emphasis Type="Italic">SST</Emphasis>) and SST Receptor 1 (<Emphasis Type="Italic">SSTR1</Emphasis>) genes that are linked with growth traits

Somatostatin (SST) and its receptors (SSTR1-5) appear to be important in central regulation of many metabolic systems that affect growth, adiposity and nutrient absorption. In this study, we investigated polymorphisms within the caprine SST and SSTR1 genes and determined their relationship with growth traits. As there were no sequence information of the caprine SST and SSTR1 genes, we explored their DNA sequence and genomic organizations. The caprine SST gene is organized in two exons and is transcribed into an mRNA containing 351 bp of sequence coding for a protein of 116 amino acids. Its protein sequences showed substantial similarity (97–99%) to its respective orthologs from cattle, human and mouse. We also cloned and sequenced a 1.2 kb DNA fragment which contained the major part of the coding region and 3′ UTR of the caprine SSTR1 gene. We then detected the polymorphisms in these determined sequences by PCR-SSCP and DNA sequencing methods in 459 goats from four breeds. Four SNPs (GU014693:g.647T>C, GU014693:g.844A>C, GU014693:g.970T>C, GU014693:g.1039T>A), segregating as two haplotypes (T-A-T-T and C-C-C-A), were identified in intron 1 of the caprine SST gene and showed the associations to body length and body height (P < 0.05). Two SNPs (GU014695:g.801 C>T, GU014695:g.948 C>T) were identified in the caprine SSTR1 gene. Significant associations between the three genotypes of GU014695:801 C>T and body length, body height, and chest circumference was observed (P < 0.05). These results suggest that the caprine SST and SSTR1 genes are strong candidate genes that influence growth traits in goat.     

Genotypes,haplotypes and diplotypes of <Emphasis Type="Italic">IGF</Emphasis>-<Emphasis Type="Italic">II</Emphasis> SNPs and their association with growth traits in largemouth bass (<Emphasis Type="Italic">Micropterus salmoides</Emphasis>)

Insulin-like growth factor II (IGF-II) is involved in the regulation of somatic growth and metabolism in many fishes. IGF-II is an important candidate gene for growth traits in fishes and its polymorphisms were associated with the growth traits. The aim of this study is to screen single nucleotide polymorphisms (SNPs) of the largemouth bass (Micropterus salmoides) IGF-II gene and to analyze potential association between IGF-II gene polymorphisms and growth traits in largemouth bass. Four SNPs (C127T, T1012G, C1836T and C1861T) were detected and verified by DNA sequencing in the largemouth bass IGF-II gene. These SNPs were found to organize into seven haplotypes, which formed 13 diplotypes (haplotype pairs). Association analysis showed that four individual SNPs were not significantly associated with growth traits. Significant associations were, however, noted between diplotypes and growth traits (P < 0.05). The fish with H1H3 (CTCC/CGCC) and H1H5 (CTCC/TTTT) had greater body weight than those with H1H1 (CTCC/CTCC), H1H2 (CTCC/TGTT) and H4H4 (TGCT/TGCT/) did. Our data suggest a significant association between genetic variations in the largemouth bass IGF-II gene and growth traits. IGF-II SNPs could be used as potential genetic markers in future breeding programs of largemouth bass.     

Analysis of genetic variability at codon 42 within caprine <Emphasis Type="Italic">prion protein</Emphasis> gene in relation to production traits in Chinese domestic breeds

In this study, genetic variability at codon 42 within prion protein (PRNP) gene and its associations with production traits were investigated in 2002 goats from four Chinese domestic breeds. The frequencies of allele “A” ranged from 0.353 to 0.562 in analyzed goat breeds with Hardy–Weinberg equilibrium (P > 0.05) except Xinong Sannen (XNSN) dairy breed. The establishment of relationships between different genotypes and growth traits was performed in Inner Mongolia white Cashmere (IMWC) breed and revealed an association of the polymorphism with body weight at 7-year-old goats (P = 0.033). The individuals with genotype GG showed heavier body weight than those with genotype AA. Moreover, association analysis detected two significant associations between different genotypes and cashmere yield and fiber length in IMWC breed (P = 0.009, P = 0.048, respectively). In addition, three significant associations of different genotypes with density of milk (a.m. and p.m.), solids-not-fat of milk (P = 0.013, P = 0.009 and P = 0.002), respectively, were found in XNSN breed. Genotype GG had better milk quality than others. These findings suggested that the polymorphism of codon 42 within PRNP was a useful DNA marker for eliminating or selecting excellent individuals in relation to production traits in marker-assist selection breeding of goat.     

Novel SNPs in the <Emphasis Type="Italic">ATP1B2</Emphasis> gene and their associations with milk yield,milk composition and heat-resistance traits in Chinese Holstein cows

Genetic association analysis was applied to examine the effect of the Na⁺/K⁺-ATPase beta 2 subunit (ATP1B2) gene on rectal temperature, milk traits, K⁺ levels and Na⁺/K⁺-ATPase (NKA) activity in the red blood cells of 1001 Chinese Holstein cows under normal and heat-stress conditions. We detected two novel single nucleotide polymorphisms, G2258A and C2833T, in the second and fourth introns, respectively, of ATP1B2. G2258A significantly affected milk fat content (P < 0.05) and 305-day milk yield (P < 0.01), but not milk protein content. C2833T significantly affected milk protein content (P < 0.01) and 305-day milk yield (P < 0.05), but not milk fat content. Calculated gene substitution effects suggested that A to G substitution in G2258A, and T to C substitution in C2833T, positively affected milk fat content, 305-day milk yield and somatic cell score, but negatively affected milk protein content. We also detected significant variation in milk fat content, milk protein content, 305-day milk yield and somatic cell scores (P < 0.05 or P < 0.01) among the nine ATP1B2 haplotypes. Under heat-stress, the C2833T polymorphism was significantly related to rectal temperature (P < 0.01), red blood cell K⁺ levels, NKA activity and milk yield (P < 0.05). Cows with the TT genotype showed the desirable characteristics of low rectal temperature and red blood cell K⁺, low decline rate in milk yield and red blood cell NKA activity. This study suggests that the ATP1B2 single nucleotide polymorphism C2833T is a genetic marker of heat-resistance traits in Chinese Holstein cows.     

Effects of <Emphasis Type="Italic">Mbo</Emphasis>II and <Emphasis Type="Italic">BspM</Emphasis>I polymorphisms in the gonadotropin releasing hormone receptor (<Emphasis Type="Italic">GnRHR</Emphasis>) gene on sperm quality in Holstein bulls

The hypothalamic gonadotropin-releasing hormone receptor (GnRHR) plays an essential physiological role in reproductive function, which triggers the synthesis and release of luteinizing hormone and follicle stimulating hormone in the pituitary. The objective of this study was to investigate the effects of polymorphisms of GnRHR gene on the quality of fresh and frozen semen in Holstein bulls. The PCR-RFLP method was applied to detect G286A and T340C transitions determining MboII and BspMI polymorphisms, respectively, in the exon I of bovine GnRHR gene and evaluated its associations with sperm quality traits in 131 Holstein bulls. In polymorphic locus 286, bulls with the GA genotype had significantly higher sperm motility in frozen semen (FMOT) than GG genotype (P < 0.01). In polymorphic locus 340, bulls with heterozygote CT genotype had significantly higher sperm motility (MOT), semen volume per ejaculate (VOL), and lower abnormal spermatozoa rate (ASR) than homozygote TT genotype (P < 0.05). Bulls contained one A allele or C allele had a favorable, positive effect on sperm quality traits. These results indicate that GnRHR gene can be a potential marker for improving sperm quality traits, and imply that bulls with GA or CT genotype should be selected in breeding program.     

Genetic mapping of the <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> avirulence gene corresponding to the <Emphasis Type="Italic">LepR1</Emphasis> resistance gene of <Emphasis Type="Italic">Brassica napus</Emphasis>

AvrLepR1 of the fungal pathogen Leptosphaeria maculans is the avirulence gene that corresponds to Brassica LepR1, a plant gene controlling dominant, race-specific resistance to this pathogen. An in vitro cross between the virulent L. maculans isolate, 87-41, and the avirulent isolate, 99-56, was performed in order to map the AvrLepR1 gene. The disease reactions of the 94 of the resulting F₁ progenies were tested on the canola line ddm-12-6s-1, which carries LepR1. There were 44 avirulent progenies and 50 virulent progenies suggesting a 1:1 segregation ratio and that the avirulence of 99-56 on ddm-12-6s-1 is controlled by a single gene. Tetrad analysis also indicated a 1:1 segregation ratio. The AvrLepR1 gene was positioned on a genetic map of L. maculans relative to 259 sequence-related amplified polymorphism (SRAP) markers, two cloned avirulence genes (AvrLm1 and AvrLm4-7) and the mating type locus (MAT1). The genetic map consisted of 36 linkage groups, ranging in size from 13.1 to 163.7 cM, and spanned a total of 2,076.4 cM. The AvrLepR1 locus was mapped to linkage group 4, in the 13.1 cM interval flanked by the SRAP markers SBG49-110 and FT161-223. The AvrLm4-7 locus was also positioned on linkage group 4, close to but distinct from the AvrLepR1 locus, in the 5.4 cM interval flanked by FT161-223 and P1314-300. This work will make possible the further characterization and map-based cloning of AvrLepR1. A combination of genetic mapping and pathogenicity tests demonstrated that AvrLepR1 is different from each of the L. maculans avirulence genes that have been characterized previously.     

Age-related macular degeneration and genetic polymorphisms of glutathione <Emphasis Type="Italic">S</Emphasis>-transferases M1 (<Emphasis Type="Italic">GSTM1</Emphasis>) and T1 (<Emphasis Type="Italic">GSTT1</Emphasis>)

The aim of this study is to understand the multifactorial causes of age-related macular degeneration (ARMD), and, therefore, it is reasonable to investigate whether genetic polymorphisms of antioxidant enzymes (GSTM1 and GSTT1) contribute to the development of ARMD. This study consisted of 112 subjects (44 females, 68 males) with exudative ARMD, who were recruited from Khalili Hospital ophthalmic clinic in Shiraz (southern Iran), referred by vitreoretinal surgeon. Also 112 sex-matched controls (44 females, 68 males) were randomly selected from unrelated volunteers in the same clinic. We excluded patients and controls with cataract or past history of cataract surgery, asthma, past history of malignancy, cardiovascular disease that on medication and known cases of glaucoma, because these traits were associated with GSTM1 and/or GSTT1 polymorphisms. There was no association between polymorphisms of neither GSTM1 nor GSTT1 and risk of ARMD. The combination genotypes of GSTM1 and GSTT1 were not associated with the risk of ARMD. We considered the time of deterioration of vision as the time of onset of exudative ARMD. The Kaplan–Meier analysis revealed that there was significant difference between genotypes of GSTM1 (log rank statistic = 7.03, df = 1, P = 0.008). The age at onset among GSTM1 null genotype was lower than the active genotype of GSTM1. Our results support the hypothesis that the protein encoded by the GSTM1 gene might have a protective function against oxidative stress in retina. Since the age at onset is influenced by the GSTM1 polymorphism, this implies that GSTM1 is a modifier gene.     

Polymorphism of <Emphasis Type="Italic">DLK1</Emphasis> and <Emphasis Type="Italic">CLPG</Emphasis> gene and their association with phenotypic traits in Chinese cattle

DLK1 and CLPG were located in DLK1-GTL2 imprinted cluster. They all affected muscle growth and meat tenderness. The functional importance of DLK1 and CLPG imply that the variation of the genes could affect the growth traits of animal. PCR-SSCP and sequencing were used to analyze the four loci of DLK1 gene and CLPG gene in 1109 individuals, which belong to eight breeds/species of bovidae, including cattle, buffalo and yak. A synonymous mutation (C451T) was detected in exon 5 of DLK1 in Qinchuan cattle, but didn’t change significantly with phenotypic traits. Three genotypes AA, AB and AC of CLPG were identified in Jiaxian cattle. The associations analyst of different genotypes showed that the individuals with genotypes AA and AC had a greater body weight and longer body length than those with genotype AB (P < 0.05 and P < 0.01, respectively); the AA individuals were different from those AB (P < 0.05) in the circumference of cannon bone. No polymorphism was observed in the other populations at other loci. These results were in agreement with the homology analysis: DLK1 and CLPG genes were in a highly conserved.     

Association of the <Emphasis Type="Italic">DGAT1</Emphasis> gene polymorphism in bulls with cow milk performance

The substitution of lysine for alanine (K232A) in the acyl-CoA:diacylglycerol acyltransferase, which is encoded by the DGAT1 gene, was tested for the significance for breeding evaluation of bulls of the holsteinized Black-and-White breed. The breeding value was estimated by the DYDC (daughter yield deviation to contemporaries) method with modification. The frequency of allele 232K in the bulls examined was 0.28, lower than in Holstein bulls (0.4–0.6). The greatest effect of the A232K substitution was observed for the percent fat milk yield (1.4σ) and milk yield (0.76σ), and a lower effect was established for the milk protein yield (0.47σ) and percent protein milk yield (0.44σ). In the case of milk fat yield, the effect was nonsignificant. A method was proposed for converting the data on fat yield in order to obtain significant results in this case as well. The effect of the A232K substitution was estimated at 154 kg for milk yield, 2.8 kg for milk protein yield, 0.079% for percent milk fat, and 0.015% for percent milk protein yield. The results are discussed in the context of multiple pleiotropic effects of the K232A substitution in the DGAT1 gene. It is proposed that the K232A substitution of the DGAT1 gene may be used as a golden standard in comparisons of the effect on milk production traits for the total gene set. This approach will allow a meta-analysis of the gene effects in spite of the different dairy cattle breeds and methods used to analyze their breeding value. In view of more than 30-year experience of using sperm of Holstein bulls, including those breeds in North America, it was noted that the effect of the A232K substitution on milk production traits agreed well with the data reported for the North American commercial population of Holstein cattle.     

Analysis of polymorphism within <Emphasis Type="Italic">POU1F1</Emphasis> gene in relation to milk production traits in dairy Sarda sheep breed

The ovine POU1F1 gene is localized on chromosome 1 and it contains five introns and six exons. In different mammalian species some mutations in different exons are associated with different production traits. The aim of our research was to study the POU1F1 gene nucleotide sequence to detect possible polymorphisms and their relationships with milk productive traits in Sarda breed sheep. The study had been conducted on 140 ewes, 4 or 5 years old coming from a farm located in Sardinia. All the animals were multiparous, lactating and in their third to fifth lactation. Individual milk yield had been recorded monthly and for each sample fat, protein, casein, lactose, and somatic cell count values were analysed. A jugular blood sample was collected from each ewe to perform genomic DNA extraction. PCR, SSCP and sequencing analysis were carried out to examine the six exons to highlight possible SNPs. One-way ANOVA was used to analyse association of variants with milk yield and/or its composition. Two novel SNP were found: 121 C>T in the 5′UTR of the fourth intron fragment and 249 G>A in the 3′UTR of the sixth exon fragment. The statistical analysis did not shown association between milk productive traits and the found polymorphisms. However, further investigations about the promoter region or the prophet genes, like the PROP-1, could clarify its exact role in regulating the productive traits in sheep.     

<Emphasis Type="Italic">ATXN</Emphasis>-<Emphasis Type="Italic">2</Emphasis> CAG repeat expansions are interrupted in ALS patients

It has recently been suggested that short expansions of CAG repeat in the gene ATXN-2 causing SCA2 (spinocerebellar ataxia type 2) are associated with an increased risk of amyotrophic lateral sclerosis (ALS) in the populations of the USA and northern Europe. In this study, we investigated the role of ATXN-2 in Italian patients clinically diagnosed with ALS and characterized the molecular structure of ATXN-2 expansions. We assessed the size of the CAG repeat in ATXN-2 exon 1 in 232 Italian ALS patients and 395 matched controls. ATXN-2 expanded alleles containing >30 repeats have been observed in seven sporadic ALS patients (3.0%), while being absent in the controls (p = 0.00089). Four out of the seven patients had an ATXN-2 allele in the intermediate-fully pathological range: one with 32 repeats, 2 with 33 repeats and 1 with 37 repeats, accounting for 1.7% of the ALS cohort. Sequencing of expanded (>32) alleles showed that they were all interrupted with at least one CAA triplet. ATXN-2 alleles with the same length and structure have been reported in SCA2 patients with parkinsonism or in familial and sporadic Parkinson. Conversely, the phenotype of the present patients was typically ALS with no signs or symptoms of ataxia or parkinsonism. In conclusion, the findings of ATXN-2 expansions in pure ALS cases suggest that ALS may be a third phenotype (alongside ataxia/parkinsonism and pure Parkinson) associated with ATXN-2 interrupted alleles.     

siRNA targeting <Emphasis Type="Italic">Notch</Emphasis>-<Emphasis Type="Italic">1</Emphasis> decreases glioma stem cell proliferation and tumor growth

Glioblastoma multiforme (GBM), the most common brain tumor in adults, is neurologically destructive and has a dismal response to virtually all therapeutic modalities. One phenomenon that can contribute to this complexity is the presence of a relatively small subset of glioma stem cells (GSCs) within the tumor and the activation of pathways that control cellular differentiation. The Notch signaling pathway, which is responsible for maintaining a balance between cell proliferation and apoptosis, is believed to be deregulated in cancer stem cells (CSCs), leading to tumor growth through the generation or expansion of CSCs. In this study, Notch-1 small interfering RNA (siRNA) was used to silence Notch-1 gene expression in GSCs. An MTT assay demonstrated inhibitory effects on the proliferation of GSCs in vitro. Real-time PCR showed that Notch-1 expression levels were markedly decreased in GSCs transfected with Notch-1 siRNA in vitro. Notch-1 silenced GSCs engrafted on Balb/c nude mice showed a significantly greater reduction in oncogenicity than the control group (P < 0.05). Furthermore, direct intratumoral injections of Notch-1-siRNA/PEI significantly delayed the growth of pre-established tumors in nude mice (P < 0.05). These results suggest that siRNA-mediated silencing of the Notch-1 gene may represent a novel target for gene therapy of GBM.