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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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Tyrosinase, encoded by TYR gene, is an enzyme that plays a major role in mammalian pigmentation. It catalyzes the oxidation of L-dihydroxy-phenylalanine (DOPA) to DOPA quinone, a precursor of both types of melanin: eumelanin and pheomelanin. TYR is commonly known as the albino locus since mutations in this gene result in albinism in several species. However, many other TYR mutations have been found to cause diluted phenotypes, like the Himalayan or chinchilla phenotypes in mice. The llama ( Lama glama ) presents a wide variety of coat colors ranging from non-diluted phenotypes (eumelanic and pheomelanic), through different degrees of dilution, to white. To investigate the possible contribution of TYR gene to coat color variation in llamas, we sequenced TYR exons and their flanking regions and genotyped animals with diluted, non-diluted, and white coat, including three blue-eyed white individuals. Moreover, we analyzed mRNA expression levels in skin biopsies by qPCR. TYR coding region presented nine SNPs, of which three were non-synonymous, c.428A > G, c.859G > T, and c.1490G > T. We also identified seven polymorphisms in non-coding regions, including two microsatellites, an homopolymeric repeat, and five SNPs: one in the promoter region (c.1-26C > T), two in the 3'-UTR, and two flanking the exons. Although no complete association was found between coat color and SNPs, c.1-26C > T was partially associated to diluted phenotypes. Additionally, the frequency of the G allele from c.428A > G was significantly higher in white compared to non-diluted. Results from qPCR showed that expression levels of TYR in white llamas were significantly lower ( p < 0.05) than those in diluted and non-diluted phenotypes. Screening for variation in regulatory regions of TYR did not reveal polymorphisms that explain such differences. However, data from this study showed that TYR expression levels play a role in llama pigmentation.

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.

South American camelids comprise the guanaco (Lama guanicoe) and the vicuña (Vicugna vicugna), which are wild species, and the domestic llama (Lama glama) and alpaca (Lama pacos). This paper presents the first complete mitochondrial (mt) genome of the guanaco and the mt coding sequence of the vicuña. The guanaco mtDNA is 16,649 nt long and its composition and organization are similar to the mitochondrial genome of other mammals. Excluding the control region, comparison of the complete guanaco and vicuña mtDNA showed 4.4% sequence divergence. Nucleotide differences in peptide coding genes varied from 1.9% in ATP6 to 6.4% in Cyt b. These values are compatible with the close relatedness of both species identified by other authors. Based on the differences between the control region sequence here reported and that previously described, we also discuss the occurrence of NUMTs in the genome of South American camelids.

We investigated the genetic differentiation of five X-chromosome STR markers among five native South American Amerindian populations inhabiting three different areas of the Gran Chaco: Mocoví, Chorote, Wichí, Lengua, and Ayoreo. The observed genetic structure showed correspondence with geographic distribution more clearly than previous information obtained from autosomal STRs for the same samples. On the other hand, X-chromosome STR data did not agree with linguistic affinities. These markers proved to be informative for the study of the native populations of the Gran Chaco region.

We report here a review of the seventh mitochondrial DNA (mtDNA) exercise undertaken by the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) corresponding to the period 2003-2004. Five reference bloodstains from five donors (M1-M5), a mixed stain of saliva and semen (M6), and a hair sample (M7) were submitted to each participating laboratory for nuclear DNA (nDNA; autosomal STR and Y-STR) and mtDNA analysis. Laboratories were asked to investigate the contributors of samples M6 and M7 among the reference donors (M1-M5). A total of 34 laboratories reported total or partial mtDNA sequence data from both, the reference bloodstains (M1-M5) and the hair sample (M7) concluding a match between mtDNA profiles of M5 and M7. Autosomal STR and Y-STR profiling was the preferred strategy to investigate the contributors of the semen/saliva mixture (M6). Nuclear DNA profiles were consistent with a mixture of saliva from the donor (female) of M4 and semen from donor M5, being the semen (XY) profile the dominant component of the mixture. Strikingly, and in contradiction to the nuclear DNA analysis, mtDNA sequencing results yield a more simple result: only the saliva contribution (M4) was detected, either after preferential lysis or after complete DNA digestion. Some labs provided with several explanations for this finding and carried out additional experiments to explain this apparent contradictory result. The results pointed to the existence of different relative amounts of nuclear and mtDNAs in saliva and semen. We conclude that this circumstance could strongly influence the interpretation of the mtDNA evidence in unbalanced mixtures and in consequence lead to false exclusions. During the GEP-ISFG annual conference a validation study was planned to progress in the interpretation of mtDNA from different mixtures.

The south American rodent Graomys griseoflavus exhibits a remarkable chromosome polymorphism as a consequence of four Robertsonian fusions. Focusing on the genetic analysis of the taxon, genome organization of all karyomorphs was studied at chromosome and molecular organization level. Cytogenetic (G, NOR and Re banding) and molecular (satellite and mitochondrial DNAs) events accompanying chromosome divergence allowed tracing a phylogenetic relationship among all karyomorphs. Available data led to propose that chromosome evolution of G. griseoflavus occurred in a non-random sequence of centric fusions, supporting the hypothesis of single origin for Robertsonian karyomorphs.

A population study for the F13B locus was carried out in a total of 396 South American individuals. The analysis comprised new data from 5 Amerindian populations, existing data from 3 Amerindian populations, and I urban sample from La Plata, Argentina. In both pooled Amerindian and La Plata samples, 6 alleles were found. The individual Amerindian samples showed a lower number of alleles, changes in modal alleles, and restricted variability. Interpopulation comparisons revealed significant differences among samples from distinct geographical regions. Differences among the groups were also corroborated by the F(ST) statistic. Data support the hypothesis that genetic drift and gene flow influence Amerindian differentiation.

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.

The restriction map of rDNA from South American camelids and the Bactrian camel was analyzed by digestion of high-molecular-weight DNA with endonucleases EcoRI,BamHI and the two combined followed by Southern blot hybridization with probes for the 18S and 28S rDNA sequences. We scored a total of 17 restriction sites, six of which were mapped conserved in all the species. The other eleven corresponded to spacer regions and revealed variations between these taxa. The study showed that the two groups differ in the length of the internal transcribed spacer. Also they showed the existence of two regions of fast evolution on the opposite termini of the external spacer. A restriction site present at low frequency in the non-transcribed spacer of guanaco and llama was the only difference encountered within the South American group.

Tandem satellite arrays and interspersed repetitive DNA components of the New World camelids guanaco, llama, alpaca, and vicuña and the Old World bactrian camel have been identified and compared. Southern hybridizations, using camel restriction fragments as probes, indicated that satellite DNAs in all camelids examined have been conserved since the last common ancestor about 5-10 MY ago. The hybridization profiles, however, varied from totally identical (MspI-sat) to highly differentiated (PstI-sat and EcoRI-sat) between Old and New World species. Repetitive DNA patterns specific of South American camelids were identified by most of the vicuña and guanaco probes and (a) llama and guanaco have undifferentiable patterns, supporting the view that the former is a domesticated form of the latter; (b) vicuña patterns were species-specific and in agreement with its position in a separate taxonomic unit; (c) the presence in alpaca of BamHI, TaqI and EcoRI patterns that are intermediate between those of the species above, suggested that the origin of the alpaca may be found in a cross-breed between the guanaco and vicuña.

Zinc finger-Y (Zfy) and zinc finger-X (Zfx) genes were analyzed by Southern blotting in male and female specimens of 10 species belonging to the oryzomyne-akodontine stock of Cricetidae rodents. DNA fragments were used as characters to construct a parsimony tree of the genes. Zfx and Zfy trees in general coincide with the evolutionary history of the taxa. Both trees show Oryzomys longicaudatus genes as the outgroup whereas Akodon xanthorrhinus genes are also distant from those of the other species. Oxymycterus rufus and Bolomys obscurus share related sequences, while genes from the other six Akodon species form a group of their own. It was found that 9 out of the 10 species analyzed show Zfy amplification in a range varying from 2 to 24 copies and with a pattern that is clade specific. The estimation of the average changes per character strongly suggests that Zfy has evolved more rapidly than Zfx; our estimates of the rate of nucleotide substitution are 4.6 times higher for Zfy than for Zfx.

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.

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.

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.

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.

The DNA composition and the in situ hybridization of satellite fractions were analysed in the New World camelids llama, alpaca, guanaco and vicuña. In the four camelid forms, it was possible to identify a similar main band DNA and five satellite fractions (I-V) with G + C base contents ranging from 32% to 66%. Satellites II-V from llama were in situ reannealed on chromosomes from the four camelid forms. The results obtained were: (a) the four satellites hybridized with regions of C-banding (centromeric regions of all chromosomes and short arms of some autosomes); (b) in general, homologous hybridizations (llama DNA versus llama chromosomes) were more efficient than heterologous reassociations; there were however three exceptions to this rule (vicuña and alpaca satellite fraction II, chromosome group B; vicuña fraction V, chromosome groups A and B); (c) X chromosomes from the four camelids had satellites III-V but lacked satellite II, (d) no satellite fraction was detected on chromosome Y. The analysis of the in situ hybridization patterns allowed to conclude that most or all C-banded chromosome regions comprise several satellite DNA fractions. It is, moreover, proposed that there is an ample interspecies variation in the number of chromosomes that cross-react with a given satellite fraction. Our data give further support to the close genomic kinship of New World camelids.