Chromosomal Location of Ribosomal DNA (rDNA), (GATA)n and (TTAGGG)n Telomeric Repeats in the Neogastropod Fasciolaria Lignaria (Mollusca: Prosobranchia)

This paper reports on a successful application of fluorescent in situhybridization (FISH) with three repetitive DNA probes (ribosomal DNA (rDNA), (GATA)_nand (TTAGGG)_n) in the chromosomes of Fasciolaria lignaria(Mollusca: Prosobranchia: Neogastropoda). rDNA FISH consistently identified four chromosome pairs per spread in the three examined specimens. The telomeric sequence (TTAGGG)_nhybridized with termini of all chromosomes. GATA FISH revealed abundant, dispersed minisatellite regions which were not associated to the XY sex-determining mechanism as indicated by the absence of a Y specific pattern of labelling. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genotype identification and assessment of genetic relationships in pearl millet [Pennisetum glaucum (L.) R. Br] using microsatellites and RAPDs

 The potential of DNA markers such as microsatellites, minisatellites and RAPDs was investigated in pearl millet [Pennisetum glaucum (L.) R. Br] with respect to their abundance and variability. Southern analysis, using 22 different di-, tri-, tetra- and penta-oligonucleotide probes and five minisatellite probes, identified (GATA)₄ as the most useful probe for the detection of multiple polymorphic fragments among pearl millet cultivars and landraces from India. The clustering patterns of pearl millet cultivars and landraces based on (GATA)₄ and RAPD (randomly amplified polymorphic DNA) markers differed. The landraces, representing eight states in India, could not be grouped based on their geographical distribution with the DNA markers. RAPD analysis revealed a high degree of genetic diversity among the cultivars and landraces employed in this study. The probability of an identical match by chance for any two genotypes using (GATA)₄ and RAPDs was 3.02×10^-20 for cultivars and 5.2×10^-9 for landraces. The microsatellite (GATA)₄ and RAPDs provide useful tools for genotype identification and for the assessment of genetic relationships in pearl millet. Received: 19 October 1997 / Accepted: 9 December 1997     

Instability of (GATA)<Subscript> n</Subscript> microsatellite loci in the parthenogenetic Caucasian rock lizard<Emphasis Type="Italic"> Darevskia unisexualis</Emphasis> (Lacertidae)

Mini- and microsatellites, comprising tandemly repeated short nucleotide sequences, are abundant dispersed repetitive elements that are ubiquitous in eukaryotic genomes. In humans and other bisexual species hypervariable mini- and microsatellite loci provide highly informative systems for monitoring of germline and somatic instability. However, little is known about the mechanisms by which these loci mutate in species that lack effective genetic recombination. Here, multilocus DNA fingerprinting was used to study M13 minisatellite and (GATA) _n microsatellite instability in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae). DNA fingerprinting of 25 parthenogenetic families, from six isolated populations in Armenia (comprising a total of 84 siblings), using the oligonucleotide (GATA)₄ as a hybridization probe, revealed mutant fingerprinting phenotypes in 13 siblings that differed from their mothers in several restriction DNA fragments. In three families (8 siblings), the mutations were present in the germline. Moreover, the mutant fingerprint phenotypes detected in siblings were also present in population DNA samples. No intrafamily variations in DNA fingerprint patterns were observed with the M13 minisatellite probe. Estimates of the mutation rate for (GATA) _n microsatellite loci in D. unisexualis showed that it was as high as that seen in some bisexual species, reaching 15% per sibling or 0.95% per microsatellite band. Furthermore, in one case, a somatic (GATA) _n microsatellite mutation was observed in an adult lizard. These findings directly demonstrate that mutations in (GATA) _n microsatellite loci comprise an important source of genetic variation in parthenogenetic populations of D. unisexualis.Communicated by G. P. Georgiev     

How is it that microsatellites and random oligonucleotides uncover DNA fingerprint patterns?

Minisatellites, microsatellites, and short random oligonucleotides all uncover highly polymorphic DNA fingerprint patterns in Southern analysis of genomic DNA that has been digested with a restriction enzyme having a 4-bp specificity. The polymorphic nature of the fragments is attributed to tandem repeat number variation of embedded minisatellite sequences. This explains why DNA fingerprint fragments are uncovered by minisatellite probes, but does not explain how it is that they are also uncovered by microsatellite and random oligonucleotide probes. To clarify this phenomenon, we sequenced a large bovine genomic BamHI restriction fragment hybridizing to the Jeffreys 33.6 minisatellite probe and consisting of small and large Sau3A-resistant subfragments. The large Sau3A subfragment was found to have a complex architecture, consisting of two different minisatellites, flanked and separated by stretches of unique DNA. The three unique sequences were characterized by sequence simplicity, that is, a higher than chance occurrence of tandem or dispersed repetition of simple sequence motifs. This complex repetitive structure explains the absence of Sau3A restriction sites in the large Sau3A subfragment, yet provides this subfragment with the ability to hybridize to a variety of probe sequences. It is proposed that a large class of interspered tracts sharing this complex yet simplified sequence structure is found in the genome. Each such tract would have a broad ability to hybridize to a variety of probes, yet would exhibit a dearth of restriction sites. For each restriction enzyme having 4-bp specificity, a subclass of such tracts, completely lacking the corresponding restriction sites, will be present. On digestion with the given restriction enzyme, each such tract would form a large fragment. The largest fragments would be those that contained one or more long minisatellite tracts. Some of these large fragments would be highly polymorphic by virtue of the included minisatellite sequences; by virtue of their complex structure, all would be capable of hybridizing to a wide variety of probes, uncovering a DNA fingerprint pattern.     

GATA repeats in the genome of Asellus aquaticus (Crustacea,Isopoda)

A 500 bp fragment of Drosophila genomic DNA containing 37 copies of the tetranucleotide GATA was used to probe, by Southern DNA blotting and in situ hybridization, two natural populations of the isopod crustacean Asellus aquaticus collected from the Sarno and Tiber rivers. This species does not have a recognizable sex chromosome pair. In a number of males from the Sarno population chromomycin A₃ staining reveals a heteromorphic chromosome pair. The heterochromosome has two blocks of heterochromatin. After digestion of genomic DNA with six restriction endonucleases and hybridization with the GATA probe, the two populations exhibit different fragment length patterns. No sex-linked pattern was observed in either population. In situ hybridization to chromosomes of males and females from the Sarno population does not reveal any sex-specific pattern of labelling and indicates a scattered distribution of GATA sequences on most chromosomes with some areas of preferential concentration. The heterochromatic arcas of the male heterochromosome are not labelled.by E.R. Schmidt     

Identification of hypervariable single locus minisatellite DNA probes in the blue tit Parus caeruleus

We report the isolation of a set of hypervariable minisatellite DNA sequences from a blue tit Parus caeruleus genomic DNA library. In our strategy, we cloned a minisatellite-rich DNA fraction into a charomid vector. The resulting cosmid library was screened with the two minisatellite DNA probes 33.6 and 33.15 for recombinants containing a minisatellite DNA insert. A total of 233 positive clones were isolated. Of 37 clones that have been analysed, nine gave polymorphic signals and can be used as single locus probes (SLPs). Four of the SLPs were investigated in more detail. The number of alleles, the heterozygosity and the mutation rate were estimated. Linkage analysis revealed that two of these loci were linked. The SLPs are of value to studies of the mating system and reproductive success in the blue tit, and may also be useful in population genetic studies.     

Genetic markers for the Booroola fecundity (Fec) gene in sheep

Animals from the Booroola line of Australian Merino sheep are characterized by a high ovulation rate that can be attributed to the presence of a codominant allele (Fec ^B).The specific function of the gene has not been identified. Effective use of the trait within the sheep breeding industry requires one or more genetic markers that can distinguish between alternative alleles at the locus Fec. With a combination of DNA minisatellite markers and polymorphic protein markers, a cluster of seven minisatellite fragments has been identified as being linked to the Fec gene and to the ovine A blood group locus. The minisatellite fragments have been derived from multilocus probes and hence cannot be used to define the chromosomal location of the Fec gene or to serve as diagnostic markers for Fec. The derivation of cloned single locus markers from the minisatellite fragments will enable finer scale mapping of the Fec and the A blood group locus in sheep.     

Isolation and characterization of a new gene, sre , which encodes a GATA-type regulatory protein that controls iron transport in Neurospora crassa

Multiple GATA factors – regulatory proteins with consensus zinc finger motifs that bind to DNA elements containing a GATA core sequence – exist in the filamentous fungus Neurospora crassa. One GATA factor, NIT2, controls nitrogen metabolism, whereas two others, WC-1 and WC-2, regulate genes responsive to blue light induction. A gene encoding a new GATA factor, named SRE, was isolated from Neurospora using a PCR-mediated method. Sequence analysis of the new GATA factor gene revealed an ORF specifying 587 amino acids, which is interrupted by two small introns. Unlike all previously known Neurospora GATA factors, which possess a single zinc-finger DNA-binding motif, SRE contains two GATA-type zinc fingers. The deduced amino acid sequence of SRE shows significant similarity to URBS1 of Ustilago and SREP of Penicillium. A loss-of-function mutation was created by the RIP procedure. Analysis of sre ⁺ and sre ⁻ strains revealed that SRE acts as a negative regulator of iron uptake in Neurospora by controlling the synthesis of siderophores. Siderophore biosynthesis is repressed by high iron concentrations in the wild-type strain but not in sre ⁻ mutant cells. The sre promoter contains a number of GATA sequences; however, expression of sre mRNA occurs in a constitutive fashion and is not regulated by the concentration of iron available to the cells. Received: 20 January 1998 / Accepted: 23 April 1998     

Simple GA C T A repeats characterize the X chromosomal heterochromatin of Microtus agrestis,European field vole (Rodentia,Cricetidae)

The sex chromosomes of Microtus agrestis are extremely large due to the accumulation of constitutive heterochromatin. We have identified two prominent satellite bands of 2.0 and 2.8 kb in length after HaeIII and HinfI restriction enzyme digestion of genomic DNA, respectively. These satellites are located on the heterochromatic long arm of the X chromosome as shown using Microtus x mouse somatic cell hybrids. By in-gel hybridization with oligonucleotide probes, the organization of the two satellites was studied: among the many copies of the simple tandem tetranucleotide repeat GATA are interspersed rare single GACA tetramers. One of the satellites also harbours related GGAT simple tandem repeats. In situ hybridizations with plasmid-carried or oligonucleotide GA _C ^T A probes show clustered silver grains on the long and short arm of the X chromosome. Interspersion of differently organized (GATA)_n elements is also demonstrable in the autosomal complement and on the Y chromosome. These results are discussed in the context of the evolution of vertebrate sex chromosomes in relation to heterochromatin and simple repetitive DNA sequences.     

PCR primed with minisatellite core sequences yields species-specific patterns and assessment of population variability in fishes of the genus Brycon

Minisatellite core sequences were used as single primers in polymerase chain reaction (PCR) to amplify genomic DNA in a way similar to the random amplified polymorphic DNA methodology. This technique, known as Directed Amplification of Minisatellite‐region DNA, was applied in order to differentiate three neotropical fish species (Brycon orbignyanus, B. microlepis and B. lundii) and to detect possible genetic variations among samples of the threatened species, B. lundii, collected in two regions with distinct environmental conditions in the area of influence of a hydroelectric dam. Most primers generated species‐specific banding patterns and high levels of intraspecific polymorphism. The genetic variation observed between the two sampling regions of B. lundii was also high enough to suggest the presence of distinct stocks of this species along the same river basin. The results demonstrated that minisatellite core sequences are potentially useful as single primers in PCR to assist in species and population identification. The observed genetic stock differentiation in B. lundii associated with ecological and demographic data constitute a crucial task to develop efficient conservation strategies in order to preserve the genetic diversity of this endangered fish species.     

Identification, isolation and characterization of canine minisatellite sequences

A Charomid ordered-array library containing a 2–16 Kb size fraction of MbeoI-digested canine genomic DNA has been screened with the Jeffreys multilocus probes, 33-6 and 33-15, to identify and isolate canine minisatellite sequences. Of the 48 positive clones identified, 7 were found to contain polymorphic mini-satellites with heterozygosities in the range 20–88%. The majority of the remainder were either monomorphic or dimorphic in the animals tested. Analysis of intrabreed variation in Bedlington Terriers using two polymorphic minisatellites has shown that a significant reduction occurs in the number of alleles seen compared to an agglomerated population sample, correlating with the high level of inbreeding within this breed. Flanking DNA sequence and partial repeat sequence is presented for the most polymorphic minisatellite thus far identified, cCfaMP5. The variable region in this mini-satellite is similar to human minisatellites which show a distinct purine or pyrimidine strand bias.     

(TG)n uncovers a sex-specific hybridization pattern in cattle

Screening of a bovine genomic library with the human minisatellite 33.6 probe uncovered a family of clones that, when used to probe Southern blots of bovine genomic DNA digested with the restriction enzyme HaeIII or MboI, revealed sexually dimorphic, but otherwise virtually monomorphic, patterns among the larger DNA fragments to which they hybridized. Characterization of one of these clones revealed that it contains different minisatellite sequences. The sexual dimorphism hybridization pattern observed with this clone was found to be due to multiple copies of two tandemly interspersed repeats: the simple sequence (TG)n and a previously undescribed 29-bp sequence. Both repeats appear to share many genomic loci including autosomal loci. In contrast, Southern analysis of AluI- or HinfI-digested bovine DNA with the (TG)n repeat used as a probe yielded substantial polymorphism. These results show that (i) different minisatellites can be found in a cluster, (ii) both simple and more complex repeated sequences other than the simple quaternary (GATA)n repeat can be sexually dimorphic, and (iii) simple repeats can reveal substantial polymorphism.     

Molecular Markers of Various Stages of Non-Small-Cell Lung Cancer

Studies performed in the recent decade in the Laboratory of the Regulation of Cell and Virus Oncogenes associated structural and functional defects of several oncogenes and tumor suppressor genes with various stages of non-small-cell lung cancer. High risk of lung cancer was established for carriers of rare alleles of the Hras1 minisatellite, the hypermethylated p16 ^INK4A promoter, and microsatellite defects in chromosome regions 3p12, 3p14.2, 3p22–24, 3p21, 3p25, 9p21, and 17p13. Analysis of the Hras1 minisatellite and microsatellites located in two minimal deletion overlap regions of 1p36 was shown to allow a more reliable prognosis in lung cancer. The results testified again that panels of molecular markers are useful for individual risk assessment, early and differential diagnosis, and prognosis in cancer.     

Microsatellite markers for the Mexican spotted owl (Strix occidentalis lucida)

A genomic cosmid library was used to develop seven highly polymorphic microsatellite markers for the Mexican spotted owl (Strix occidentalis lucida). These are the first reported microsatellite markers derived from this species. The cloned and sequenced repeat motifs include a triplet repeat of (AAT)_n, two tetranucleotide repeats of (GATA)_n, a tetranucleotide repeat of (ATCC)_n, a compound repeat of (GA)_n(GATA)_n and the two pentanucleotide repeats (AGAAT)_n and (ATTTT)_n. The microsatellites described represent six presumably independent loci with the two pentanucleotide repeats having originated from a single cosmid. Primer pairs allow locus‐specific amplification of each marker from Mexican spotted owl genomic DNA.     

Minisatellite polymorphisms of the SLC6A19: susceptibility in hypertension

The SLC6A19 is a human homolog of B⁰AT1 that encodes a neutral amino acid transporter. We examined the distribution of VNTR (variable number of tandem repeats; minisatellites) and conducted polymorphic analysis of SCL6A19 isolated from the genomic DNA of controls and multi-generational families. The SLC6A19 was found to contain seven blocks of minisatellites, 3 of which (SLC6A19-MS1, -MS4, and -MS7) showed polymorphism and were found to be transmitted through meiosis following Mendelian inheritance in seven families. These minisatellite polymorphisms may be useful markers for paternity mapping and DNA fingerprinting. Furthermore, we conducted a case-control study in which genomic DNA from 400 controls and 205 cases with essential hypertension was compared. A statistically significant association was identified between rare SLC6A19-MS7 alleles and the occurrence of hypertension (odds ratio, 7.87; 95% confidence interval, 0.88-70.66; and p = 0.028). These findings suggest that the rare SLC6A19-MS7 allele may be a risk factor for hypertension.     

Construction of a bacterial artificial chromosome library containing large Eco RI and Hin dIII genomic fragments of lettuce

 Existing bacterial artificial chromosome (BAC) vectors were modified to have unique EcoRI cloning sites. This provided an additional site for generating representative libraries from genomic DNA digested with a variety of enzymes. A BAC library of lettuce was constructed following the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC library of over 50,000 clones, representing one to two genome equivalents, was constructed from six ligations; average insert sizes for each ligation varied between 92.5 and 142 kb with a combined average insert size of 111 kb. The library was screened with markers linked to disease resistance genes; this identified 134 BAC clones from four regions containing resistance genes. Hybridization with low-copy genomic sequences linked to resistance genes detected fewer clones than expected from previous estimates of genome size. The lack of hybridization to chloroplast and mitochondrial sequences demonstrated that the library was predominantly composed of nuclear DNA. The unique EcoRI site in the BAC vector should allow the integration of BAC cloning with other technologies that utilize EcoRI digestion, such as AFLP^TM markers and RecA-assisted restriction endonuclease (RARE) cleavage, to clone specific large EcoRI fragments from genomic DNA. Received: 5 August 1996 / Accepted: 23 August 1996     

Nucleotide sequence of a highly repeated DNA sequence and its chromosomal localization in Allium fistulosum

A highly repeated DNA sequence with a repeating unit of approximately 380bp was found in EcoRV digests of the total genomic DNA of Allium fistulosum. Three independent clones containing this unit were isolated, and their repeating units sequenced. These units showed more than 94% sequence homology, and the copy number was estimated to be about 2.8×10⁶ per haploid genome. In situ hybridization, with the repeating unit as a probe, and C-banding analyses indicated that the repeated DNA sequence of A. fistulosum is closely associated with the major C-heterochromatin in the terminal regions of all 16 chromosomes at mitotic metaphase. The characters of the repeating unit are similar to those of the A. cepa unit, which is taxonomically closely related to A. fistulosum.     

The contribution of short repeats of low sequence complexity to large conifer genomes

The abundance and genomic organization of six simple sequence repeats, consisting of di-, tri-, and tetranucleotide sequence motifs, and a minisatellite repeat have been analyzed in different gymnosperms by Southern hybridization. Within the gymnosperm genomes investigated, the abundance and genomic organization of micro- and minisatellite repeats largely follows taxonomic groupings. We found that only particular simple sequence repeat motifs are amplified in gymnosperm genomes, while others such as (CAC)₅ and (GACA)₄ are present in only low copy numbers. The variation in abundance of simple sequence motifs reflects a similar situation to that found in angiosperms. Species of the two- and three-needle pine section Pinus are relatively conserved and can be distinguished from Pinus strobus which belongs to the five-needle pine section Strobus. The hybridization pattern of Picea species, bald cypress and gingko were different from the patterns detected in the Pinus species. Furthermore, sequences with homology to the plant telomeric repeat (TTTAGGG)_n have been analyzed in the same set of gymnosperms. Telomere-like repeats are highly amplified within two- and three- needle pine genomes, such as slash pine (Pinus elliottii Engelm. var. elliottii), compared to P. strobus, Picea species, bald cypress and gingko. P. elliottii var. elliottii was used as a representative species to investigate the chromosomal organization of telomere-like sequences by fluorescence in situ hybridization (FISH). The telomere-like sequences are not restricted to the ends of chromosomes; they form large intercalary and pericentric blocks showing that they are a repeated component of the slash pine genome.Conifers have genomes larger than 20000 Mbp, and our results clearly demonstrate that repeats of low sequence complexity, such to (CA)₈, (GA)₈, (GGAT)₄ and (GATA)₄, and minisatellite- and telomere-like sequences represent a large fraction of the repetitive DNA of these species. The striking differences in abundance and genome organization of the various repeat motifs suggest that these repetitive sequences evolved differently in the gymnosperm genomes investigated. Received: 1 October 1999 / Accepted: 3 November 1999     

Mutations at the human minisatellite MS32 integrated in yeast occur with high frequency in meiosis and involve complex recombination events

Minisatellites are composed of tandem repetitive DNA sequences and are present at many positions in the human genome. They frequently mutate to new length alleles in the germline, by complex and incompletely understood recombination mechanisms which may operate during meiosis. In several minisatellites the mutation events are restricted to one end of the repeat array, indicating a possible association with elements that act in cis. Mutant alleles do not show exchange of flanking regions. To construct a model system suitable for further investigations of the mutation process, we have integrated the human minisatellite MS32, flanked by synthetic markers, in the vicinity of a meiotic recombination hot spot upstream of the LEU2 locus in the yeast Saccharomyces cerevisiae. Here we provide direct evidence for a meiotic origin of MS32 mutations. Mutation events were polarised towards both ends of the minisatellite and varied from simple duplications and deletions to complex intra- and interallelic events. Interallelic events were frequently accompanied by exchange of regions flanking the minisatellite. The results also support the notion that cis-acting elements are involved in the mutational process. The fact that MS32 mutant structures are similar in yeast and human shows that meiotic recombination plays a crucial role in both organisms and emphasises the usefulness of yeast strains harbouring minisatellites as a model system for the study of minisatellite mutation. Received: 1 March 1997 / Accepted: 16 May 1997     

The genes coding for tRNATyr of Drosophila melanogaster: Localization and determination of the gene numbers

Transfer RNA^Tyr (anticodon GA) was isolated from Drosophila melanogaster by means of Sepharose 4B, RPC-5, and polyacrylamide gel electrophoresis. The tRNA was iodinated in vitro with Na¹²⁵I and hybridized in situ to salivary gland chromosomes from Drosophila. The genes of tRNA^Tyr were localized in eight regions of the genome by autoradiography. Restriction enzyme analysis of genomic DNA indicated that the haploid Drosophila genome codes for about 23 tRNA^Tyr genes. The regions 22F and 85A each contain four to five tRNA^Tyr genes, whereas the regions 28C, 41AB, 42A, 42E, and 56D each contain two to three tRNA^Tyr genes.