Restriction Enzyme Banding Research Articles

Differentiation between Bacillus amyloliquefaciens and Bacillus subtilis isolated from a South African sugarcane processing factory using ARDRA and rpoB gene sequencing.

A total of 104 exopolysaccharide (gum)-producing bacteria were isolated from the juice screen and juice tank in a sugarcane processing factory at times of low- and high dextran concentrations in the produced sugar. Dextran is an indicator of cane deterioration and sucrose loss after harvesting of the cane. The isolates were identified as Bacillus amyloliquefaciens (96 isolates) and Bacillus subtilis (eight isolates) based on restriction enzyme banding patterns of amplified 16S rRNA genes and rpoB gene sequence analysis. Exopolysaccharide production in sugarcane is normally associated with dextran produced by Leuconostoc mesenteroides. B. amyloliquefaciens, and to a lesser extent B. subtilis, could, however, also be responsible for exopolysaccharide (slime or gum) production in cane processing factories.

Identification and characterization of phages parasitic on bradyrhizobia nodulating groundnut (Arachis hypogaea L.) in South Africa

In this study, three lytic phages (namely, PRSA-1, PRSA-2 and PRSA-26) were isolated and characterized for their morphology, host range, profile and restriction endonuclease banding pattern of genome size. The susceptible rhizobial isolates were identified by nifH and glnII sequence analysis. The results showed that all phages had polyhedral head with non-contractile tail which confirmed their relationship with the Siphoviridae family. All the three phages produced highly distinct plaques on their host bradyrhizobial lawn, and were highly sensitive to chloroform. The phage genome sizes ranged from 34.7 to 53.1 kbp. The phages were tested against groundnut-nodulating bradyrhizobial strains TUTAHSA75, TUTAHSA155 and TUTAHSA126 isolated from South African soils. The results revealed different bacterial susceptibilities to phages. Bradyrhizobial isolate TUTAHSA126 was susceptible to all three phages (i.e. PRSA-1, PRSA-2 and PRSA-26), TUTAHSA155 to two phages (i.e. PRSA-1, PRSA-2), and TUTAHSA75 to only one phage (i.e. PRSA-1). Phylogenetic analysis of nifH and glnII gene sequences of the phage-susceptible bradyrhizobial isolates revealed their close relatedness to a diverse group of Bradyrhizobium species. Phage PRSA-1 could parasitize on all three bradyrhizobial strains, which indicates its potential role in horizontal gene transfer through lysogenic conversion, and/or genetic transduction in soil microbial environments.

Cytogenetic Characterization of the Dwarf OysterOstrea stentina(Mollusca: Bivalvia) and Comparative Karyological Analysis within Ostreinae

ABSTRACT Regardless of the high economic value and large geographical distribution of oysters, the current knowledge of oyster taxonomic relationships and systematics is still limited, particularly for flat oysters. In this study, the molecular cytogenetic characterization of mitotic chromosomes of the Provence flat oyster or dwarf oyster, Ostrea stentina, was performed through Giemsa staining, chromosome measurements, in situ restriction endonuclease banding, C-banding, fluorescence in situ hybridization with major ribosomal RNA genes (ITS1), and telomeric sequence (TTAGGG)n. The karyotype (2n = 20) consisted of 6 metacentric (1, 3, 4, 6, 8, and 10) and 4 submetacentric (2, 5, 7, and 9) chromosome pairs. Chromosome treatment with HaeIII produced specific banding patterns for all chromosomal pairs, confirming the efficiency of this restriction enzyme for chromosome banding in oysters. Results for C-banding revealed the presence of heterochromatin in the telomeric regions of the short arms: on a large meta...

Localization and Characterization of the 5S rDNA Bearing Chromosome in Two Steindachneridion Species by Means of Different Cytogenetic Techniques

Karyotype macrostructure and nucleolar organizer regions have often been studied in Neotropical siluriform fishes, but data about structural rearrangements and gene mapping are scarce. 5S rDNA is localized and characterized in Sorubiminae species (Steindachneridion melanodermatum and S. scriptum) using fluorescence in situ hybridization, C- and G-banding and restriction enzyme banding (AluI, BamHI and EcoRI). Chromosome mapping of 5S rDNA genes showed only one site located on the short arms of subtelocentric chromosome pair (chromosome 23) in both species, with no apparent polymorphisms. This region is C-positive and shows a clear G-band, having no cleavage sites for the restriction enzymes tested. These genes are not syntenical with the 18S ones and their karyotype location remains conserved in the species studied, appearing as a cytogenetic marker.

Clinical features and natural history of neuroferritinopathy caused by the 458dupA FTL mutation

Sir, In 2007, we described the clinical features and natural history of neuroferritinopathy in 41 patients defined at the molecular genetic level by the presence of Eco N1 restriction digestion site in exon 4 of FTL (also referred to as FTL1 ), the gene coding for the ferritin light polypeptide (Chinnery et al. , 2007). Three French patients in our paper (Cases 13, 36 and 39 in Table 1) had an identical restriction enzyme banding pattern to the original Cumbrian neuroferritinopathy family (Curtis et al. , 2001). Their early clinical course (Caparros-Lefebvre et al. , 1997) and molecular genetic analysis (Chinnery et al. , 2003) had been previously described. We recently received a DNA sample to confirm the diagnosis in a new member of the same French family. Given the recent description of three new FTL mutations (Vidal et al. , 2004; Maciel et al. , 2005; Mancuso et al. , 2005; Ohta et al. , 2008), we now sequence exon 4 of FTL to provide a more comprehensive molecular diagnostic approach. This showed that the new French patient had a different mutation, 458dupA, which segregated with the phenotype in the French family and was not found in 300 European controls. This mutation creates the same Eco N1 restriction site as 460dupA described in the original neuroferritinopathy family (Curtis et al. , 2001), and given that both the French and English pedigrees shared a …

Supernumerary chromosomes on Southern European populations of the cockle Cerastoderma edule: Consequence of environmental pollution?

Cerastoderma edule (Cardiidae) has a diploid chromosome number of 2 n = 38, its karyotype consisting of 12 submetacentric, 4 subtelocentric and 3 telocentric chromosome pairs. Hyperdiploid cells had previously been observed in two populations of the Northern Galician coasts (northwest of Spain). The supernumerary chromosomes being easily distinguished by their reduced differentiated size and by their intra- and inter-individual variability. After the recent observation of 35% of cells with supernumerary chromosomes in a population of the Southern Galician coasts (Vigo) and 15% of cells with supernumerary chromosomes in a population of the south of Portugal (Ria Formosa, Algarve), we attempted, in this paper, an elucidation of the nature of these supernumerary chromosomes, by differential banding technique with restriction enzymes on these hyperdiploid cells. Analysis of the restriction enzyme banding of the 2 n > 38 karyotypes led us to propose the occurrence of a chromosomal fission event involving the largest submetacentric chromosome pair. This study represents the first description of the occurrence of a possible chromosomal fission in marine bivalves. Different levels of environmental pollution are suggested as possible explanation for the differences observed on the proportion of hyperdiploid cells between the Southern Portugal population and the three Galician ones.

Heterogeneity of heterochromatin in six species ofCtenomys (Rodentia: Octodontoidea: Ctenomyidae) from Argentina revealed by a combined analysis of C- and RE-banding

Exceptional chromosomal variability makesCtenomys an excellent model for evolutionary cytogenetic analysis. Six species belonging to three evolutionary lineages were studied by means of restriction endonuclease and C-chromosome banding. The resulting banding patterns were used for comparative analysis of heterochromatin distribution on chromosomes. This combined analysis allowed intra- and inter-specific heterochromatin variability to be detected, groups of species belonging to different lineages to be characterized, and phylogenetic relationships hypothesized from other data to be supported. The “ancestral group”,Ctenomys pundti andC. talarum, share three types of heterochromatin, the most abundant of which was also found in C. aff.C. opimus, suggesting that the latter species also belongs to the “ancestral group”. Additionally, within the subspeciesC. t. talarum, putative chromosomal rearrangements distinguishing two of the three chromosomal races were identified. Two species belong to an “eastern lineage”,C. osvaldoreigi andC. rosendopascuali, and share only one type of heterochromatin homogeneously distributed across their karyotypes.C. latro, the only analyzed species from the “chacoan” lineage, showed three types of heterochromatin, one of them being that which characterizes the “eastern lineage”.C. aff.C. opimus, because of its low heterochromatin content, is the most primitive karyotype of the genus yet described. The heterochromatin variability showed by these species, reflecting the evolutionary divergence toward different heterochromatin types, may have diverged since the origin of the genus. Heterochromatin amplification is proposed as a trend withinCtenomys, occurring independently of chromosomal change in diploid numbers.

C, Q, and restriction enzyme banding of the chromosomes in brook trout (Salvelinus fontinalis) and Arctic charr (Salvelinus alpinus)

The brook trout (Salvelinus fontinalis) and the Arctic charr (Salvelinus alpinus) represent different phylogenetic lines of the salmonid fish genus Salvelinus. Chromosome banding studies utilizing C, Q, and restriction enzymes reveal differences between the two species for the amount and sequence composition of their heterochromatic DNA. Arctic charr, the more recently evolved of the two species, possesses more C and Q bands than the brook trout. With the exception of Alu I, the banding patterns produced by restriction enzymes bear a close resemblance to the C band pattern. Alu I eliminates staining from large pericentromeric regions, including the centromeric heterochromatin, in the chromosomes of both species and from some telomeric regions in Arctic charr. These findings show that the evolutionary divergence of the Arctic charr from the brook trout has been accompanied by accumulation of heterochromatic DNA and subsequent sequence divergence.

Analyses of the Structure of NORs in Two Species of South American Sorubiminae Fishes (Siluriformes) by Means of Several Cytogenetic Techniques

Several cytogenetic techniques (AgNOR, C- G- and RE bandings, DAPI, CMA3 and FISH) were applied in order to analyze the structure and variability of NORs in the fish species Steindachneridion melanodermatum and S. scripta. Ag-NORs were observed on the short arm of the first acrocentric chromosome pair, coincidentally with a strong C-positive band on a large secondary constriction. In addition, NORs showed bright fluorescent signals when stained with CMA3 and treated for FISH with rDNA 18S. However, they showed negative coloration after G- and restriction enzyme banding and DAPI staining. The results evidence a substantial size polymorphism in these regions. The NOR bearing chromosomes in both species may be considered homologues because they maintain conserved characteristics, such as being interspersed with a GC-rich heterochromatin and possessing target sequences for AluI, BamHI and EcoRI.

RESTRICTION ENZYME DIGESTION CHROMOSOME BANDING ON TWO COMMERCIALLY IMPORTANT VENERID BIVALVE SPECIES: CERASTODERMA EDULE

Reliable banding techniques are a major necessity for the genetic research in marine bivalves. Restriction enzyme banding (HaeIII) was performed, in this study, on chromosomes of two commercially important species of veneroid bivalves: the clam Ruditapes decussatus (Adams and Reeve) and the cockle Cerastoderma edule. Identification of the nineteen individual chromosome pairs was obtained for both species. The cytogenetic studies made in marine molluscs have recently experienced a very fast development caused by the introduction of new molecular techniques mainly fluorescence in situ hybridization (FISH). Recently it has been shown in mammalian chromosomes that restriction enzyme banding is compatible with FISH, allowing simultaneous banding, and consequent accurate identification of the localization of the probes and unambiguously identification of the chromosome(s) carrier(s). As far as we know this is the first RE-banding obtained in karyotypes of veneroid species. The application of restriction enzyme chromosome banding in veneroids are diverse and this study can constitute a fundamental step for future gene mapping on this commercially important group of bivalves and could offer a new approach to specific problems in veneroid taxonomy and genetics.

Heteromorphic sex chromosome system with an exceptionally large Y chromosome in a catfish Steindachneridion sp. (Pimelodidae)

The chromosomes and banding patterns of Steindachneridion sp., a large catfish (Pimelodidae), endemic to the Iguaçu River, Brazil, were analyzed using conventional (C-, G-banding) and restriction enzyme banding methods. The same diploid number (2n = 56) as in other members of the genus and the family was found but the karyotype displayed an XX/XY sex chromosome system. The X chromosome was the smallest submetacentric, while the Y was the largest chromosome in the karyotype. Meiotic analysis showed 27 autosomal bivalents plus one heteromorphic XY bivalent during spermatogenesis. Sex chromosomes had no particular pattern after C-banding but G- and restriction enzyme bandings showed specific banding characteristics. The present finding represents the first report of a well-differentiated and uncommon sex chromosome system in the catfish family Pimelodidae.

Molecular and cytogenetic characterization of Crassostrea angulata chromosomes

We have conducted a molecular cytogenetic study of Crassostrea angulata, by treating its mitotic chromosomes with C-banding, fluorochrome and oligopeptide staining, restriction enzyme banding and fluorescent in situ hybridization, using three repetitive or multicopy DNAs (the GATA sequence, the TTAGGG telomeric repeat and the 5S rDNA). Results on C-banding indicate the presence of heterochromatin in several chromosomes occupying telomeric positions. The staining with DAPI, DA/DAPI and AMD/DAPI allow concluding that large regions rich in AT in the chromosomes of C. angulata do not exist. Restriction banding has shown few restriction sites for AluI (AGTC site) and a relative abundance of G-C in terminal and interstitial chromosome regions, inferred by the digestion with HaeIII (GGCC site) and BamHI (GGATCC site). In situ hybridization with GATA indicated that these repeated sequences are widely dispersed in the genome of this species, whereas hybridization with the telomeric repeat revealed small bright hybridization signals, uniform in size and intensity, on each telomere of all chromosomes but not in interstitial positions. Location of 5S rDNA displayed the presence of two 5S-bearing chromosome pairs, of large size, on the subterminal position of the karyotype of C. angulata, this location being different from the ones encoding the major ribosomal genes in chromosome pair 10 (described in a previous paper).

Restriction enzyme digestion chromosome banding in Crassostrea and Ostrea species: comparative karyological analysis within Ostreidae

Reliable banding techniques are a major necessity for genetic research in oysters. In this study, we carried out the cytogenetic characterization of four oyster species (family Ostreidae) using restriction endonuclease treatments. Chromosomes were treated with three different restriction enzymes, stained with Giemsa, and examined for banding patterns. The following species were studied: Crassostrea gigas (2n = 20; total number of bands with ApaI, 74; HaeIII, 61; PstI, 76), Crassostrea angulata (2n = 20; ApaI, 62; HaeIII, 61; PstI, 55) (subfamily Crassostreinae), Ostrea edulis (2n = 20; ApaI, 82; HaeIII, 59; PstI, 66), and Ostrea conchaphila (2n = 20; ApaI, 68; HaeIII, 62; PstI, 69) (subfamily Ostreinae). Treatment of samples with ApaI, HaeIII, and PstI produced specific banding patterns, which demonstrates the potential of these enzymes for chromosome banding in oysters. This is of special interest, since it has been recently shown in mammalian chromosomes that restriction enzyme banding is compatible with fluorescence in situ hybridization. This study therefore provides a fundamental step in genome mapping of oysters, since chromosome banding with restriction enzymes facilitates physical gene mapping in these important aquaculture species. The analysis of the banded karyotypes revealed a greater similarity within the genera of Crassostrea and Ostrea than between them.

Heterochromatin heterogeneity revealed by restriction endonuclease digestion and subsequent C-banding on bovine metaphase chromosomes.

Conventional alkaline C-banding was found to be an effective additional step in restriction endonuclease banding to improve staining contrast and to reveal hidden changes in the heterochromatic blocks. Examples of these cases are demonstrated on the bovine chromosome set using Hinf I and Taq I endonucleases in the digestion experiments.

Characterization of restriction enzyme banding polymorphisms in human chromosomes.

C and Re-banding chromosome heteromorphisms were analysed in blood cultures from 43 normal individuals. Restriction enzymes used were AluI, DdeI, HaeIII, and MboI. Chromosome pairs exhibiting heteromorphisms were: 1, 3, 4, 6, 9, 10, 12-16, and 18-22. Each individual showed a specific combination of C- and Re-banding heteromorphisms not shared by any other individual in the series. Some polymorphisms could be detected by all the banding methods used. Others could be detected by some of the banding methods, and in some cases by only one of the banding methods used. The efficiency of each banding method to detect chromosomal polymorphisms depended on the type of polymorphism and the chromosomal pair analysed. Our results indicate that Re-banding polymorphisms occur due to changes in base composition of different fractions of heterochromatin or due to the presence or absence of different heterochromatic subsets. C- and Re-banding are complementary methods that expand the identification of chromosomal markers and which can be used to identify the parental origin of individual chromosomes.

Cytogenetic Analysis of A- and B-Chromosomes of Rhamdia hilarii (Teleostei, Pimelodidae): C-Banding, Silver Nitrate and CMA3 Staining and Restriction Endonuclease Banding.

We investigated the chromosome complement of Rhamdia hilarii using C-banding, silver nitrate and CMA3 staining, and chromosome digestion with 9 restriction endonucleases (RE). The results revealed that this species has 2n=58 to 2n=60 chromosomes, with the numerical variation being due to the presence of 1 or 2 metacentric B-chromosomes. The C-banding pattern showed a small amount of heterochromatin in A-chromosomes and heterochromatin in both telomeric regions of B-chromosomes. The Ag-NOR and CMA3 staining revealed that the rDNA loci were located in the terminal position on the short arm of a pair of submetacentric chromosomes. The heterochromatin of the B-chromosomes was not stained brightly with CMA3 showing that these regions segments are not GC-rich. Chromosome digestion with different REs permitted the identification of homologous chromosome pairs in some cases. On the basis of the results, we discuss some aspects of chromosome structure and the origin of the B-chromosomes in Rhamdia.

Cytogenetic analysis of A- and B-chromosomes of Prochilodus lineatus (Teleostei, Prochilodontidae) using different restriction enzyme banding and staining methods.

Different cytogenetic techniques were used to analyse the chromosomes of Prochilodus lineatus with the main objective of comparing the base composition of A- and B-chromosomes. The results of digestion of chromosomes with 10 different restriction endonucleases (REs), silver staining, CMA3 staining and C-banding indicated the existence of different classes of highly repetitive DNA in the A-set and also suggested the existence of compositional differences between the chromatin of A- and B-chromosomes. The 5-BrdU incorporation technique showed a late replicating pattern in all B-chromosomes and in some heterochromatic pericentromeric regions of A-chromosomes. The cleavage with RE BamHI produced a band pattern in all chromosomes of P. lineatus which permitted the tentative pairing of homologues in the karyotype of this species. We concluded that the combined use of the above techniques can contribute to the correct identification of chromosomes and the karyotypic analysis in fishes. On the basis of the results, some aspects of chromosome structure and the origin of the B-chromosomes in P. lineatus are discussed.

Centromeric heterochromatin in the cattle rob(1;29) translocation: alpha-satellite I sequences, in-situ MspI digestion patterns, chromomycin staining and C-bands.

The centromeric regions and alpha-satellite I sequence were studied on chromosomes 1, 29 and the rob(1;29) translocation in a Portuguese breed of cattle, Barrosa, carrying the translocation. Rob(1;29) centromeric regions showed heterochromatic bands with propidium iodide but, unlike the acrocentric autosomes, no strong centromeric bands were revealed with chromomycin A3. An alpha-satellite I sequence was not found at the centromeres of the X, Y and rob(1;29) chromosomes in the breed, although it was present at the centromeres of all acrocentric chromosomes including 1 and 29. Restriction enzyme banding with MspI revealed polymorphisms between different rob(1;29) chromosomes in both centromeric and intercalary regions. The data show that the centromeric region of the rob(1;29) chromosome has lost the alpha-satellite I sequences, while retaining other heterochromatin, and suggest that this important and widespread translocation has occurred multiple times.

Karyotypic analysis of the cotton boll weevil, Anthonomus grandis Boheman.

The diploid chromosome number of the cotton boll weevil, Anthonomus grandis Boheman, is 44. Both C- and N-banding techniques of mitotic cells demonstrated constitutive heterochromatin in the p arm of the eight largest chromosomes, the p arm of the X chromosome, and the centromeric region of autosomal groups A-D. Neither the y nor the group E autosomes appeared to contain constitutive heterochromatin. Supernumerary chromosomes were not found in the boll weevil. Restriction endonuclease banding of primary spermatocytes revealed a rod-shaped Xy tetrad in which the X and y were terminally associated. The p arm of the large, submetacentric X was C-band positive. While two of the autosomal tetrads were typically ring-shaped in primary spermatocytes, the remaining 19 autosomal tetrads were rod-shaped.

The karyotype of the Iberian imperial eagle (Aquila adalberti) analyzed by classical and DNA replication banding

We report here for the first time the karyotype of the Iberian imperial eagle (Aquila adalberti). All eagles examined had a diploid number of 82 chromosomes and a greater number of microchromosomes (12 pairs) than has been found in all other species of the Accipitridae family. This karyotypic evidence corroborates the recent separation of A. adalberti from A. heliaca on the basis of molecular data. RB-FPG banding induced a specific banding pattern that allowed us to identify homologous chromosome pairs and revealed features about late and early replicating regions. Several chromosome banding techniques (C-, CMA₃-, and restriction endonuclease banding and silver staining) were used to characterize the karyotype more accurately. Two GC-rich, late-replicating heterochromatin regions were found in the W chromosome. These regions are AluI resistant and can be used for sex determination in this species. All microchromosomes were heterochromatic, GC rich, and late replicating. Silver staining revealed active nucleolus organizing regions on a pair of microchromosomes that were entirely heterochromatic and stained intensely after CMA₃-banding. Different chromosome rearrangements are discussed in order to establish the phylogenetic relationship between A. adalberti and its most closely related species, A. heliaca.