Isolation and characterization of 5S rDNA sequences in catfishes genome (Heptapteridae and Pseudopimelodidae): perspectives for rDNA studies in fish by C0t method

Abstract

Sequences of 5S ribosomal RNA (rRNA) are extensively used in fish cytogenomic studies, once they have a flexible organization at the chromosomal level, showing inter-and intra-specific variation in number and position in karyotypes. Sequences from the genome of Imparfinis schubarti (Heptapteridae) were isolated, aiming to understand the organization of 5S rDNA families in the fish genome. The isolation of 5S rDNA from the genome of I. schubarti was carried out by reassociation kinetics (C 0 t) and PCR amplification. The obtained sequences were cloned for the construction of a micro-library. The obtained clones were sequenced and hybridized in I. schubarti and Microglanis cottoides (Pseudopimelodidae) for chromosome mapping. An analysis of the sequence alignments with other fish groups was accomplished.Both methods were effective when using 5S rDNA for hybridization in I. schubarti genome. However, the C 0 t method enabled the use of a complete 5S rRNA gene, which was also successful in the hybridization of M. cottoides. Nevertheless, this gene was obtained only partially by PCR. The hybridization results and sequence analyses showed that intact 5S regions are more appropriate for the probe operation, due to conserved structure and motifs. This study contributes to a better understanding of the organization of multigene families in catfish's genomes.

Genetic and Chromosomal Differentiation ofRhamdia quelen(Siluriformes, Heptapteridae) Revealed by Repetitive Molecular Markers and DNA Barcoding

“…20 Preparation and labeling of probes for fluorescence in situ hybridization Probes for 18S rDNA of Prochilodus argenteus (Hatanaka and Galetti 21 ) and U2 snRNA of Eigenmannia virescens used in the double fluorescence in situ hybridization (FISH) were labeled by polymerase chain reaction (PCR) with biotin-16-dUTP and the signal detected with avidin-FITC (Life Technologies, Carlsbad, CA), and were labeled with digoxigenin-11-dUTP and the signal detected with anti-digoxigenin-rhodamine (Roche Applied Science, Indianapolis, IN), respectively. The 5S rDNA probe in Imparfinis schubarti (Gouveia et al 22 ) was labeled by PCR with the nucleotide digoxigenin-11-dUTP 1 mM, and the signal was detected with anti-digoxigenin-rhodamine (Roche Applied Science). FISH was conducted according to the methodology by Pinkel et al 23 Fiber stretch for fiber-FISH Cytogenetic preparations using a large number of interphase nuclei were conducted as described by de Barros et al 24 Cell suspensions were dripped onto glass slides and washed in 1 • PBS for 2 min at room temperature (25°C).…”

Cytogenetic Trends in Two Families of the Neotropical Catfishes: Heptapteridae and Pseudopimelodidae (Siluriformes)

“…The location and number of the rDNA 5S are relatively variable occupying preferentially interstitial regions on one chromosome pair or two chromosome pairs (Table 1), in some cases syntenic with regions rDNA 18S, as already described by other authors. 11,16,25,[32][33][34] Gouveia et al 25,33 isolated 5S rDNA sequences and transposon (Tc1-mariner) from I. schubarti genome and found that transposable elements may be acting along these sequences, showing hybridization in some species of Heptapteridae and Pseudopimelodidae, and seem to be related to the movement of 5S rDNA genes in these fish genomes. Recently, Souza-Shibatta et al 35 reevaluated M. cottoides systematics, using morphological and molecular data, with sequence analysis of the COI (cytochrome oxidase I) gene.…”

“…and 5S rDNA of I. schubarti was isolated as described by Gouveia et al25 Probes were labeled with DIG-Nick Translation Kit (Roche Applied Science, Mannheim, Germany) cat number 11745816910 or BioNickÔ Labeling System Kit (Invitrogen Life Technology, Carlsbad) cat number 18247015. Preparations were covered with 50 lL of hybridization mixture containing 100 ng of labelled probe (7.5 lL), 50% formamide (30 lL), dextran sulphate 50% (12 lL) and 20 • saline-sodium citrate (SSC) (10.5 lL).…”