A single nucleotide polymorphism in an exon dictates allele dependent differential splicing of episialin mRNA

The episialin gene (MUC1) encodes an epithelial mucin containing a variable number of repeats with a length of twenty amino acids, resulting in many different alleles that can be subdivided into two size classes. The episialin pre-mRNA uses either one of two neighbouring splice acceptor sites for exon 2, which mainly encodes the repeats. Using the genetic polymorphism of the episialin gene to identify different alleles, we show here that the splice site recognition is allele dependent and is based on a single A/G nucleotide difference in exon 2 eight nucleotides downstream of the second splice acceptor site. Transfection experiments confirm that this polymorphic nucleotide regulates the splice site selection. The identity of this nucleotide is in most cases correlated with one of the size classes of the alleles, indicating that mutations altering the number of repeats seldom arise by unequal cross-over between the repeat regions.     

PCR-RFLPs within the lactate dehydrogenase (LDH-A) gene of the domestic pigeon (Columba livia var. domestica)

A total of 45 racing pigeons were genotyped using PCR-RFLP method. PCR product of the LDH-A gene was amplified according to the Long-PCR procedure. The amplification products were digested with restriction enzymes. PCR-RFLPs for two restriction enzymes, HaeIII and NlaIV, were observed. Two pairs of alleles LDH-A(A) and LDH-A(B) for LDH-A-NlaIV polymorphism and LDH-A(C) and LDH-A(D) for LDH-A-HaeIII polymorphism were detected in the homozygous and heterozygous states. Frequencies of alleles were as follows: A - 0.622, B - 0.378 and C - 0.256, D - 0.744.     

Allelic variation in the intron 2 and 3 of the MICA gene.

We have determined by sequencing the allelic variation in intron 2 and 3 of hte MICA gene for a total of 22 different alleles. Sequencing of introns was performed in two directions, using DNA from homozygous cell lines from families and from unrelated individuals. Intron 2 is 273 bp long and did in the alleles investigated not reveal any length polymorphism. We found a total of eight polymorphic positions which exhibit a strict biallelism, as it is also found in the polymorphisms for exon 2, 3 and 4 of MICA. Intron 3 is 586 bp long an required an additional set of primers placed near the middle of this intron in order to allow a complete bidirectional sequence. In intron 3, a total of 10 polymorphic positions were identified. Interestingly, we found two variants of the allele MICA*002 which are distinguished only by one basepair difference in intron 3. The variant MICA*002A is associated with HLA-B35 and B58, while the allele MICA*002B is associated with B38 and B39.     

Recombination and gene conversion-like events may contribute to ABO gene diversity causing various phenotypes

We identified five different alleles, tentatively named ABO*O301, *0302, *R102, *R103, and *A110, in Japanese individuals possessing the blood group O phenotype. These alleles lack the guanine deletion at nucleotide position 261 which is shared by a majority of O alleles. Nucleotide sequence analysis revealed that *0301 and *0302 had single nonsynonymous substitutions compared with *A101 or *A102 responsible for the A1 phenotype. Analysis of intron 6 at the ABO gene by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing revealed that *R102 and *R103 had chimeric sequences of A-02 and B-02, respectively, from exons 6 to 7. In the analysis of five other chimeric alleles detected in the same manner, we identified a total of four different recombination-breakpoints within or near intron 6. When 510 unrelated Japanese were examined, the frequency of the chimeric alleles generated by recombination in intron 6 or exon 7 was estimated to be 1.7%. In addition, we found that *O301, *A110, *C101, *A111, and 35% of *A102 had a unique A-B-A chimeric sequence at intron 6, presumed to originate from a gene conversion-like event. We had previously established that *A110 also had an A-O2-A chimeric sequence around nucleotide position 646 in exon 7. Thus this allele has an A-B-A-O2-A chimeric sequence from intron 6 to exon 7 probably generated by two different gene conversions. Similar patchwork sequences around nucleotide position 646 in exon 7 were observed in two other new alleles responsible for the Ax and B3 phenotypes. Thus, the site is presumably a hotspot for gene conversion. These results indicate that both recombination and gene conversion-like events play important roles in generating ABO gene diversity.     

Genetic polymorphism of the kappa-casein gene in Brazilian cattle

Frequencies of kappa-casein gene alleles were determined in 1316 animals from the Brazilian Bos indicus genetic groups (Sindhi cows, Gyr sires, Gyr cows, Guzerat sires, Guzerat cows, Nellore sires, and Gyr x Holstein crossbreds) by means of polymerase chain reaction-restriction fragment length polymorphism analysis using two independent restriction nucleases (Hinf I and HaeIII). The genotyping of kappa-casein alleles (A and B) is of practical importance, since the B allele is found to correlate with commercially valuable parameters of cheese yielding efficiency. The frequencies of the B allele of kappa-casein among breeds ranged from 0.01 to 0.30. The Sindhi breed had the highest frequency for the B allele (0.30), while the frequencies of this allele in other breeds ranged from 0.01 to 0.18. A wide variation in the B allele frequency among B. indicus breeds was found suggesting that molecular selection for animals carrying the B allele could impact breeding programs for dairy production.     

Genetic enhancement of RNA-processing defects by a dominant mutation in B52, the Drosophila gene for an SR protein splicing factor.

SR proteins are essential for pre-mRNA splicing in vitro, act early in the splicing pathway, and can influence alternative splice site choice. Here we describe the isolation of both dominant and loss-of-function alleles of B52, the gene for a Drosophila SR protein. The allele B52ED was identified as a dominant second-site enhancer of white-apricot (wa), a retrotransposon insertion in the second intron of the eye pigmentation gene white with a complex RNA-processing defect. B52ED also exaggerates the mutant phenotype of a distinct white allele carrying a 5' splice site mutation (wDR18), and alters the pattern of sex-specific splicing at doublesex under sensitized conditions, so that the male-specific splice is favored. In addition to being a dominant enhancer of these RNA-processing defects, B52ED is a recessive lethal allele that fails to complement other lethal alleles of B52. Comparison of B52ED with the B52+ allele from which it was derived revealed a single change in a conserved amino acid in the beta 4 strand of the first RNA-binding domain of B52, which suggests that altered RNA binding is responsible for the dominant phenotype. Reversion of the B52ED dominant allele with X rays led to the isolation of a B52 null allele. Together, these results indicate a critical role for the SR protein B52 in pre-mRNA splicing in vivo.     

Polymorphisms of the insulin-like growth factor-binding protein 3 gene (IGFBP3) in gayal (Bos frontalis)

The gene coding for insulin-like growth factor-binding protein 3 (IGFBP3) is important for regulation of growth, development and metabolism in mammals. The present investigation was conducted to study nucleotide polymorphism of the IGFBP3 in gayal (Bos frontalis) and to compare the variations with those which occur in other ruminants. A fragment of 645 base pairs of the IGFBP3 covering a part of exon 2, the complete intron 2 and exon 3 and a part of intron 3 was amplified, sequenced (n=46) and digested (n=79) with HaeIII restriction enzyme from 125 collected gayal samples. Nine single nucleotide polymorphisms (SNPs) [C14T, A122C, C137T, G144C, C155T, G213A, C279A, G334A and G460A] were identified and located in intron 2, revealing high genetic variability. The alignment of nucleotide sequences was found to be very similar to those for other bovid species. Sequencing and HaeIII digestion showed that frequency of alleles C and A [consisting of fragments of sizes 56, 64, 228, 264, 282, 298 and 497 bp (CC genotype)] was 0.96 and 0.04 for the SNP C279A. Moreover, the genotype frequency of the SNP C279A in gayal was compared with that in other ruminants and it appears that this polymorphism may be associated with low fat content and rapid growth in this rare species.     

A 5' intronic splice site polymorphism leads to a null allele of the P2X7 gene in 1-2% of the Caucasian population

The P2X(7) gene is important for the innate immune response but known polymorphisms do not explain all subjects with loss of P2X(7) function. A splice site mutation (g-->t) was found at position +1 of the first intron of the P2X(7) gene in 7 of 336 Caucasians and 1 of 39 subjects of Indian ethnicity. All eight subjects were heterozygous for the uncommon 1513A-->C polymorphism of the P2X(7) gene. RT-PCR and sequencing showed the splice site mutation was on the 1513C allele in the Caucasians and on the 1513A allele in the Indian subject. The splice site mutation is an inherited polymorphism and gives rise to a P2X(7) null allele in 1-2% of the Caucasian population.     

Identification of a splice-site mutation in the aldolase B gene from an individual with hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is a potentially fatal autosomal recessive disease of carbohydrate metabolism. HFI patients exhibit a deficiency of fructose 1-phosphate aldolase (aldolase B), the isozyme expressed in tissues that metabolize fructose. The eight protein-coding exons, including splicing signals, of the aldolase B gene from one HFI patient were amplified by PCR. Dot-blot hybridization of the amplified DNA with allele-specific oligonucleotide (ASO) probes revealed a previously described A149P mutation in one allele from the proband. The mutation in the other allele was identified by direct sequencing of the double-stranded PCR-amplified material from the proband. The nucleotide sequence of exon 9 revealed a 7-base deletion/1-base insertion (delta 7 + 1) at the 3' splice site of intron 8 in one allele. This mutation was confirmed by cloning PCR-amplified exon 9 of the proband and determining the sequence of each allele separately. ASO analysis of 18 family members confirmed the Mendelian inheritance of both mutant alleles. The implications of this unique splice-site mutation in HFI are discussed.     

Homing of a group II intron from Lactococcus lactis subsp. lactis ML3.

Ll.ltrB is a functional group II intron located within a gene (ltrB) encoding a conjugative relaxase essential for transfer of the lactococcal element pRSO1. In this work, the Ll.ltrB intron was shown to be an independent mobile element capable of inserting into an intronless allele of the ltrB gene. Ll.ltrB was not observed to insert into a deletion derivative of the ltrB gene in which the intron splice site was removed. In contrast, a second vector containing a 271-nucleotide segment of ltrB spanning the Ll.ltrB splice site was shown to be a proficient recipient of intron insertion. Efficient homing was observed in the absence of a functional host homologous recombination system. This work demonstrates that the Ll.ltrB intron is a novel site-specific mobile element in lactococci and that group II intron self-transfer is a mechanism for intron dissemination among bacteria.     

Conserved signals around the 5' splice sites in eukaryotic nuclear precursor mRNAs: G-runs are frequent in the introns and C in the exons near both 5' and 3' splice sites

It is known that the GT doublet is well conserved at the 5' exon/intron splice junction and is frequently embedded in the AGGT quartet. Although only the underlined G is invariable, splicing and ligation are accurately executed. In this work we search for additional conserved potential signals which may aid in 5' splice site recognition. Extensive searches which are not limited to a preconceived consensus sequence are carried out. We investigate the distributions of the 256 quartets in a 1000 nucleotide span around the 5' splice sites in approximately 1700 eukaryotic nuclear precursor mRNAs. Several potential signals are noted. Of particular interest are quartets containing runs of G, e.g., G4, G3T, G3C, G3A and AG3 in the intron immediately downstream and some C-containing quartets in the exon upstream of the 5' splice site. In an analogous calculation, (A)GGG(A) has also been found to be frequent in the intron, 60 nucleotides upstream and (A)CCC(A) in the exon downstream of the 3' splice site. These results are consistent with the recent indications that exon sequences may play a role in efficient splicing. Some models are proposed.     

Identification and Characterization of CD300H,a New Member of the Human CD300 Immunoreceptor Family

Recruitment of circulating monocytes and neutrophils to infection sites is essential for host defense against infections. Here, we identified a previously unannotated gene that encodes an immunoglobulin-like receptor, designated CD300H, which is located in the CD300 gene cluster. CD300H has a short cytoplasmic tail and associates with the signaling adaptor proteins, DAP12 and DAP10. CD300H is expressed on CD16⁺ monocytes and myeloid dendritic cells. Ligation of CD300H on CD16⁺ monocytes and myeloid dendritic cells with anti-CD300H monoclonal antibody induced the production of neutrophil chemoattractants. Interestingly, CD300H expression varied among healthy subjects, who could be classified into two groups according to “positive” and “negative” expression. Genomic sequence analysis revealed a single-nucleotide substitution (rs905709 (G→A)) at a splice donor site on intron 1 on either one or both alleles. The International HapMap Project database has demonstrated that homozygosity for the A allele of single nucleotide polymorphism (SNP) rs905709 (“negative” expression) is highly frequent in Han Chinese in Beijing, Japanese in Tokyo, and Europeans (A/A genotype frequencies 0.349, 0.167, and 0.138, respectively) but extremely rare in Sub-Saharan African populations. Together, these results suggest that CD300H may play an important role in innate immunity, at least in populations that carry the G/G or G/A genotype of CD300H.     

Single nucleotide polymorphisms (SNPs) within Bov-A2 SINE in the second intron of bovine and buffalo k-casein (CSN3) gene

The genetic polymorphism of an entire Bov-A2 element located in the second intron of the buffalo and bovine k-casein (CSN3) gene was investigated by amplification and sequencing of PCR products. Single nucleotide polymorphisms were detected. A PCR-RFLP method was developed to detect an A or G mutation at position 605 of bovine Bov-A2 element which creates a BfaI polymorphic site. The frequencies of the B allele, with the BfaI site, were for 0.275, 0.775, 0.750, 0.975, respectively, for Italian Holstein Friesian, Grey Alpine, Friuli Red Pied and Reggio bovine breeds. The mutation rate (substitutions and deletions/insertions per nucleotide site per year) was 2.5 x 10(-9) for Bov-A2 sequences in the second intron of CSN3. The comparison with other Bov-A2 elements suggests that this retroelement might be an important source of single nucleotide polymorphism for analysis of Bovidae genomes.     

The role of branchpoint-3'' splice site spacing and interaction between intron terminal nucleotides in 3'' splice site selection in Saccharomyces cerevisiae.

A conserved 3' splice site YAG is essential for the second step of pre-mRNA splicing but no trans-acting factor recognizing this sequence has been found. A direct, non-Watson-Crick interaction between the intron terminal nucleotides was suggested to affect YAG selection. The mechanism of YAG recognition was proposed to involve 5' to 3' scanning originating from the branchpoint or the polypyrimidine tract. We have constructed a yeast intron harbouring two closely spaced 3' splice sites. Preferential selection of a wild-type site over mutant ones indicated that the two sites are competing. For two identical sequences, the proximal site is selected. As previously observed, an A at the first intron nucleotide spliced most efficiently with a 3' splice site UAC. In this context, UAA or UAU were also more efficient 3' splice sites than UAG and competed more efficiently than the wild-type sequence with a 3' splice site UAC. We observed that a U at the first intron nucleotide is used for splicing in combination with 3' splice sites UAG, UAA or UAU. Our data indicate that the 3' splice site is not primarily selected through an interaction with the first intron nucleotide. Selection of the 3' splice site depends critically on its distance from the branchpoint but does not occur by a simple leaky scanning mechanism.     

Functionally distinct nucleic acid binding sites for a group I intron encoded RNA maturase/DNA homing endonuclease

A large number of group I introns encode a family of homologous proteins that either promote intron splicing (maturases) or are site-specific DNA endonucleases that function in intron mobility (a process called "homing"). Genetic studies have shown that some of these proteins have both activities, yet how a single protein carries out both functions remains obscure. The similarity between respective DNA-binding sites and the RNA structure near the 5' and 3' splice sites has fueled speculation that such proteins may use analogous interactions to perform both functions. The Aspergillus nidulans mitochondrial COB group I intron encodes a bi-functional protein, I-AniI, that has both RNA maturase and site-specific DNA endonuclease activities in vitro. Here, we show that I-AniI shows distinctive features of the endonuclease family to which it belongs, including highly specific, tight binding and sequential DNA strand cleavage. Competition experiments demonstrate that I-AniI binds the COB intron RNA even in saturating concentrations of its DNA target site substrate, suggesting that the protein has a separate binding site for RNA. In addition, we provide evidence that two different DNA-binding site mutants of I-AniI have little effect on the protein's RNA maturation activity. Since RNA splicing is likely a secondary adaptation of the protein, these observations support a model in which homing endonucleases may have developed maturase function by utilizing a hitherto "non-functional" protein surface.     

Tyrosinase and tyrosinase related protein 1 alleles specify domestic cat coat color phenotypes of the albino and brown loci

The genes encoding enzymes of the tyrosinase family are strong candidates for coat color variation in mammals. To investigate their influence in domestic cat coat color, we determined the complete nucleotide coding sequence of the domestic cat genes tyrosinase (TYR)--a plausible candidate gene for the albino (C) locus, and tyrosinase related protein 1 (TYRP1)--a candidate gene for the brown (B) locus. Sequence variants between individuals exhibiting variation in pigmentation were submitted to association studies. In TYR, two nonsynonymous substitutions encoding TYR-G301R and TYR-G227W were associated with the siamese and burmese phenotypes of the albino locus, respectively. TYRP1 was mapped on chromosome D4 within 5 cM of a highly polymorphic microsatellite, previously found to be fixed in a cat breed selected for the chocolate (b) allele of the B locus, which reinforced TYRP1 as a candidate gene for the B locus in the domestic cat. Two DNA polymorphisms, one leading to a TYRP1-A3G substitution in the signal peptide and another to an in-frame insertion TYRP1-421ins17/18 caused by a donor splice site mutation in intron 6, were associated with the chocolate (b) allele. A premature UAG stop codon at position 100 of TYRP1 was associated with a second allele of the B locus, cinnamon (b(l)). The results provide very strong evidence that the specific nucleotide variants of feline TYR (chromosome D1) are causative of the siamese (c(s)) and burmese (c(b)) alleles of the albino locus, as well as nucleotide variants of TYRP1 (chromosome D4) as specifying the chocolate (b) and cinnamon (b(l)) alleles of the B locus.