Characterization and expression analysis of KIT and MITF-M genes in llamas and their relation to white coat color.

The llama (Lama glama) is a fiber-producing species that presents a wide range of coat colors, among which white is one of the most important for the textile industry. However, there is little information about the molecular mechanisms that control the white phenotype in this species. In domestic mammals, a white coat is usually produced by mutations in the KIT proto-oncogene receptor tyrosine kinase (KIT) and microphthalmia-associated transcription factor (MITF) genes. In this work we have sequenced and described the coding regions of KIT and MITF-M, the melanocyte-specific isoform, and the two transcriptional variants MITF-M(-) and MITF-M(+). Moreover, we studied the expression of these genes in the skin of white and colored llamas. Although no variants were revealed to be associated with white coat color, significant differences between phenotypes were observed in the expression levels of KIT and MITF-M. Interestingly, white llamas expressed less MITF-M(+) than did colored ones, which is consistent with a consequent reduction in the synthesis of melanin. Even though our results indicate that downregulation of KIT and MITF-M expression is involved in white phenotype production in llamas, the causative gene of white coat color remains unknown.

Molecular characterization of the llama FGF5 gene and identification of putative loss of function mutations.

Llama, the most numerous domestic camelid in Argentina, has good fiber-production ability. Although a few genes related to other productive traits have been characterized, the molecular genetic basis of fiber growth control in camelids is still poorly understood. Fibroblast growth factor 5 (FGF5) is a secreted signaling protein that controls hair growth in humans and other mammals. Mutations in the FGF5 gene have been associated with long-hair phenotypes in several species. Here, we sequenced the llama FGF5 gene, which consists of three exons encoding 813 bp. cDNA analysis from hair follicles revealed the expression of two FGF5 alternative spliced transcripts, in one of which exon 2 is absent. DNA variation analysis showed four polymorphisms in the coding region: a synonymous SNP (c.210A>G), a single base deletion (c.348delA), a 12-bp insertion (c.351_352insCATATAACATAG) and a non-sense mutation (c.499C>T). The deletion was always found together with the insertion forming a haplotype and producing a putative truncated protein of 123 amino acids. The c.499C>T mutation also leads to a premature stop codon at position 168. In both cases, critical functional domains of FGF5, including one heparin binding site, are lost. All animals analyzed were homozygous for one of the deleterious mutations or compound heterozygous for both (i.e. c.348delA, c.351_352insCATATAACATAG/c.499T). Sequencing of guanaco samples showed that the FGF5 gene encodes a full-length 270-amino acid protein. These results suggest that FGF5 is likely functional in short-haired wild species and non-functional in the domestic fiber-producing species, the llama.

Genetic diversity and conservation status of managed vicuña (Vicugna vicugna) populations in Argentina.

The vicuña (Vicugna vicugna) was indiscriminately hunted for more than 400 years and, by the end of 1960s, it was seriously endangered. At that time, a captive breeding program was initiated in Argentina by the National Institute of Agricultural Technology (INTA) with the aim of preserving the species. Nowadays, vicuñas are managed in captivity and in the wild to obtain their valuable fiber. The current genetic status of Argentinean vicuña populations is virtually unknown. Using mitochondrial DNA and microsatellite markers, we assessed levels of genetic diversity of vicuña populations managed in the wild and compared it with a captive population from INTA. Furthermore, we examined levels of genetic structure and evidence for historical bottlenecks. Overall, all populations revealed high genetic variability with no signs of inbreeding. Levels of genetic diversity between captive and wild populations were not significantly different, although the captive population showed the lowest estimates of allelic richness, number of mitochondrial haplotypes, and haplotype diversity. Significant genetic differentiation at microsatellite markers was found between free-living populations from Jujuy and Catamarca provinces. Moreover, microsatellite data also revealed genetic structure within the Catamarca management area. Genetic signatures of past bottlenecks were detected in wild populations by the Garza Williamson test. Results from this study are discussed in relation to the conservation and management of the species.

The ASIP gene in the llama (Lama glama): Alternative transcripts, expression and relation with color phenotypes.

The Agouti gene (ASIP) is one of the most important genes for coat color determination in mammals. It has a complex structure with several promoters and alternative non-coding first exons that are transcribed into mRNAs with different 5'UTR. These mRNA isoforms regulate the temporal and spatial expression of the gene, producing diverse pigmentation patterns. Here, we studied ASIP transcriptional variants and their expression in the skin of llamas with different coat color phenotypes. We also described the ASIP locus, including promoter usage and the splicing events that originate each transcript variant. Using 5'RACE-PCR we isolated seven ASIP transcripts with alternative 5'UTR, where exons 1A, 1A', 1C, 1D, and a novel non-coding exon 1A" were identified. Additionally, new alternative spliced forms were found. The diversity of ASIP 5'UTRs is originated by a complex pattern of alternative promoter usage, multiple transcription start sites and splicing events that include exon skipping and alternative 3' splicing site selection. We found that ASIP was highly expressed in llamas with white and brown phenotypes while black animals presented very low expression. The main responsible for this difference was a fusion transcript between ASIP and NCOA6 genes, which was present in the skin of white and brown llamas but not in the black ones. The rest of ASIP transcripts presented very low expression in the skin, indicating that the main regulation point for ASIP gene expression is at the transcriptional level. Nevertheless, the characteristics of the 5'UTRs sequences suggest that alternative transcripts could be regulated differently at the protein synthesis level.

Identification of camelid specific residues in mitochondrial ATP synthase subunits.

ATP synthase is an enzyme involved in oxidative phosphorylation from prokaryotic to eukaryotic cells. In mammals it comprises at least 16 subunits from which the mitochondrial encoded ATP6 and ATP8 are essential. Mitochondrial genes variations have been suggested to allow rapid human and animal adaptation to new climates and dietary conditions (Mishmar et al. 2003). Camelidae taxa are uniquely adapted to extremely hot and dry climates of African-Asian territories and to cold and hypoxic environments of the South American Andean region. We sequenced and analyzed ATP6 and ATP8 genes in all camelid species. Based on the available structural data and evolutionary conservation of the deduced proteins we identified features proper of the group. In Old World camels the ATP8, important in the assembly of the F0 complex, showed a number of positively charged residues higher than in the other aligned species. In ATP6 we found the camelid specific substitutions Q47H and I106V that occur in sites highly conserved in other species. We speculate that these changes may have functional importance.

Effect of the metal chelating agent o-phenanthroline on the DNA and chromosome damage induced by bleomycin in Chinese hamster ovary cells.

A 30-min pulse treatment with bleomycin to Chinese hamster ovary cells in culture produces DNA degradation and chromosomal aberrations in a dose-dependent manner. Bleomycin also induces a long-lasting effect on the cell cycle producing a lengthening of two or more cycles after the treatment. The presence of o-phenanthroline, which chelates metal ions, totally inhibits DNA cleavage and the appearance of chromosome aberrations while partially correcting the lengthening of the cell cycle. These findings suggest that an important cellular target for bleomycin is the DNA. Chromosomal aberrations are a secondary effect resulting from DNA cleavage. On the other hand, the increase in the duration of the cell cycle is probably induced by DNA degradation and, perhaps, by damage to other cellular structures.

Mutation rates at Y chromosome specific microsatellites.

A collaborative work was carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG) to estimate Y-STR mutation rates. Seventeen Y chromosome STR loci (DYS19, DYS385, DYS389I and II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS460, DYS461, DYS635 [GATA C4], GATA H4, and GATA A10) were analyzed in a sample of 3,026 father/son pairs. Among 27,029 allele transfers, 54 mutations were observed, with an overall mutation rate across the 17 loci of 1.998 x 10(-3) (95% CI, 1.501 x 10(-3) to 2.606 x 10(-3)). With just one exception, all of the mutations were single-step, and they were observed only once per gametogenesis. Repeat gains were more frequent than losses, longer alleles were found to be more mutable, and the mutation rate seemed to increase with the father's age. Hum Mutat 26(6), 520-528, 2005. (c) 2005 Wiley-Liss, Inc.

Allele frequencies of six STR loci in Argentine populations.

Allele frequencies of six short tandem repeat (STR) loci were determined in a Caucasian urban sample of La Plata city and three Amerindian sample populations of Argentina. Allele frequencies showed differences between urbans and Amerindians, and among Amerindians as well. The degree of genetic differentiation of subpopulations was mainly due to the Amerindian contribution. Mapuche, Mocovi, and pooled Amerindian populations showed little evidence of HW disequilibrium, and association of alleles. In the urban sample, there is no evidence of population substructuring. Forensic probabilities of exclusion and matching showed high differences between the population groups. Finally, La Plata sample did not show differences with Caucasians from other geographic regions.

South American camelid illegal traffic detection by means of molecular markers.

South American camelids comprise the wild species guanaco and vicuña and their respective domestic relatives llama and alpaca. The aim of the present study was to determine by DNA analysis to which of these species belong a herd of camelids confiscated from a llama breeder but alleged to be alpacas by the prosecution, and to evaluate the usefulness of mitochondrial and autosomal DNA markers to solve judicial cases involving camelid taxa. Cytochrome b and cytochrome oxidase I mitochondrial genes and 7 STR were analyzed in 25 confiscated samples. Mitochondrial results were inconclusive because 18 of the sequestered samples presented haplotypes that corresponded to the guanaco haplogroup and the remaining seven belonged to a vicuña linage. Microsatellite data of casework samples and llama reference samples revealed different genetic profiles by the presence of private alleles at two microsatellites suggesting that the confiscated animals could be alpaca, or at least alpaca hybrids instead of pure llama.

2006 GEP-ISFG collaborative exercise on mtDNA: reflections about interpretation, artefacts, and DNA mixtures.

We report the results of the seventh edition of the GEP-ISFG mitochondrial DNA (mtDNA) collaborative exercise. The samples submitted to the participant laboratories were blood stains from a maternity case and simulated forensic samples, including a case of mixture. The success rate for the blood stains was moderate ( approximately 77%); even though four inexperienced laboratories concentrated about one-third of the total errors. A similar success was obtained for the analysis of mixed samples (78.8% for a hair-saliva mixture and 69.2% for a saliva-saliva mixture). Two laboratories also dissected the haplotypes contributing to the saliva-saliva mixture. Most of the errors were due to reading problems and misinterpretation of electropherograms, demonstrating once more that the lack of a solid devised experimental approach is the main cause of error in mtDNA testing.

Genetic diversity and differentiation of guanaco populations from Argentina inferred from microsatellite data.

Genotype data from 14 microsatellite markers were used to assess the genetic diversity and differentiation of four guanaco populations from Argentine Patagonia. These animals were recently captured in the wild and maintained in semi-captivity for fibre production. Considerable genetic diversity in these populations was suggested by the finding of a total of 162 alleles, an average mean number of alleles per locus ranging from 6.50 to 8.19, and H(e) values ranging from 0.66 to 0.74. Assessment of population differentiation showed moderate but significant values of F(ST)=0.071 (P=0.000) and R(ST)=0.083 (P=0.000). An amova test showed that the genetic variation among populations was 5.6% while within populations it was 94.4%. A number of 6.6 migrants per generation may support these results. Unambiguous individual assignment to original populations was obtained for the Pilcaniyeu, Las Heras and La Esperanza populations. The erroneous assignment of 18.75% Rio Mayo individuals to the Las Heras population can be explained by the low genetic differentiation found between these two populations. Thirty-nine of 56 loci per population combinations were in Hardy--Weinberg disequilibrium because of guanaco heterozygote deficiency, which may be explained by population subdivision. The high level of genetic diversity of the guanacos analysed here indicates that the Patagonian guanaco constitutes an important genetic resource for conservation or economic utilization programmes.

Molecular analysis of chromosomal polymorphism in the South American cricetid, Graomys griseoflavus.

Graomys griseoflavus is a South American phyllotine rodent widespread in Argentina that shows a high frequency of Robertsonian fusions (RFs). DNA restriction with EcoRI produced a 250-bp repeated family (EG250) specific for the genus. Southern hybridization and sequencing analysis indicate that the EG250 family is heterogeneous, comprising at least two subfamilies. In situ hybridized EG250 probe showed a centromere location in almost all chromosomes. In all karyomorphs C-banding was negative, but restriction enzyme banding (Re-banding) with Alul and Mbol showed centromeric blocks in the autosomes that will generate Robertsonian fusions. Thus, we found three groups of chromosomes: (a) EG250 and Re-banding negative; (b) EG250 positive and Re-banding negative; and (c) EG250 and Re-banding positive. We consider that group (b) is more the result of chromatin condensation state than that of the frequency of recognition sites for the enzymes used. Restriction enzyme blocks would appear in regions with heterochromatic EG250 subfamilies, while lack of banding would be due to decondensed EG250 subfamilies becoming an easier target for chromosomal restriction. It is suggested that heterochromatic EG250 DNA provides a favourable molecular environment for Robertsonian fusion occurrence.

Ag-NOR staining and in situ hybridization of rDNA in the chromosomes of the South American camelids.

The location and frequency of Ag-stained NORs and sites of rDNA hybridization were studied in the chromosomes of the South American camelids. In the four camelids these regions occur distally on chromosomes 18, 21, and 27 and the smallest biarmed elements. Quantitative analysis of NOR distribution showed variations between both cells and species. In llama, guanaco and alpaca the NORs number averaged 6 per cell, this being higher than in vicuña where the average was 3. Relative frequencies of NOR-bearing chromosomes in the four camelids were similar. Yet, in vicuña virtual absence of NOR sites on one of the smallest biarmed pairs was observed. The rDNA sites assessed in llama and vicuña by in situ hybridization with cloned 18S DNA were coincident with the NOR locations and with the frequencies characteristics for each species. Moreover, varying the exposure time of the autoradiographs, labeling patterns specific for each camelid were observed. Grain counts on individual chromosomes indicated that under our conditions one month exposure is enough to demonstrate all the rDNA sites available in the complement of llama. Conversely, at least two months are necessary to show the total sites existing in vicuña. Most probably this finding reflects the presence of variations in the amount of copies of the ribosomal genes per chromosome.

DNA of Akodon (Rodentia, Cricetidae). I. Biophysical characterization.

Buoyant density in CsCl, melting temperature, and G + C base content of the DNA from four species of Akodon (Rodentia, Cricetidae) were determined. The buoyant density values of 1.699-1.701 g/cm3 were in accordance with the data reported for other cricetids. No satellite bands were seen in neutral CsCl. The Tm values determined in 1 x SSC ranged from 86.2 to 87.0 C, which corresponds to G + C contents of 41.2-43.2%. There was good agreement in DNA base composition of the four species, although values were slightly higher in A. obscurus, suggesting a certain degree of interspecies variability.

Liver chromatin fractions in Mus and Akodon. The concept of constitutive heterochromatin.

The liver chromatin from Mus musculus and Akodon molinae was separated in 8 fractions by differential centrifugation. Like fractions from both species showed approximately similar contents of DNA, equivalent ratios of histone to non-histone proteins, corresponding template activities and equal amounts of positive C-banded material. On the other hand, heavy chromatin fractions of Mus were highly enriched in satellite DNA whereas no satellite DNA was found in Akodon chromatin. Heavy chromatin fractions isolated by differential sedimentation have been usually homologued with the constitutive heterochromatin. The properties of the constitutive chromatin are discussed and the validity of the foregoing concept is challenged. It is proposed to define the constitutive heterochromatin as those chromatin regions comprising highly repeated DNA sequences clustered in restricted areas of chromosomes and not transcribed (satellite DNA).

Direct visualization of the sites of DNA methylation in human, and mosquito chromosomes.

Human and mosquito fixed chromosomes were digested with restriction endonucleases that are inhibited by the presence of 5-methylcytosine in their restriction sites (Hha I, Hin PI, Hpa II), and with endonucleases for which cleavage is less dependent on the state of methylation (Taq I, Msp I). Methylation-dependent enzymes extracted low DNA amounts from human chromosomes, while methylation-independent enzymes extracted moderate to high amounts of DNA. After DNA demethylation with 5-azacytidine the isoschizomers Hpa II (methylation-dependent) and Msp I (methylation-independent) extracted 12-fold and 1.4-fold amounts of DNA from human chromosomes, respectively. These findings indicate that human DNA has a high concentration of Hpa II and Msp I restriction sites (CCGG), and that the internal C of this sequence is methylated in most cases, while the external cytosine is methylated less often. All the enzymes tested released moderate amounts of DNA from mosquito chromosomes whether or not the DNA was demethylated with 5-azacytidine. Hpa II induced banding in the centromere chromosome regions. After demethylation with 5-azacytidine this banding disappeared. Mosquito DNA has therefore, moderate to high frequencies of nonmethylated CpG duplets. The only exception is the centromeric DNA, in which the high levels of C methylation present produce cleavage by Hpa II and the appearance of banding. Centromere regions of human chromosomes 1 have a moderately low concentration of Hpa II-Msp I restriction sites.