Incomplete EcoRI digestion may lead to false diagnosis of fragile X syndrome

Molecular diagnosis of fragile X syndrome is usually performed using Southern blot analysis of DNA digested with EcoRI. In the course of diagnostic studies, we observed that a specific EcoRI restriction site in the fragile X gene (FMR1) is sometimes refractory to digestion, generating additional fragments on a Southern blot suggestive of a full mutation in FMR1. This may lead to a false-positive diagnosis of fragile X syndrome. Such additional bands are avoided by the use of HindIII instead of EcoRI. Therefore, we recommend the use of HindIII for the molecular diagnosis of fragile X syndrome. Received: 11 September 1997 / Accepted: 25 September 1997     

Restriction fragment length polymorphism of DQB and DRB class II genes of the ovine major histocompatibility complex

The ovine major histocompatibility complex (MhcOvar) class II region was investigated by Southern blot hybridizations using ovine probes specific for the second exons of Ovar-DRB and Ovar-DQB genes. Multiple bands were revealed when genomic DNA was digested with each of five restriction enzymes (Bam HI, Eco RI, Hin dIII, PvuII and TaqI), and successively hybridized with the two radiolabeled ovine probes. Restriction fragment length polymorphisms (RFLPs) were analysed in 89 sheep originating from six inbred families and the inheritance of the fragment patterns was determined. Forty-one fragments were recorded with the DQB probe; 32 were detected with the DRB probe. They constituted 9 DQB and 10 DRB allelic patterns. Twelve DQB-DRB haplotypes were resolved in this study.     

Organization of the interferon-inducible 2′,5′-oligoadenylate-dependent ribonuclease L (RNase L) gene of mouse

Interferons (IFNs) induce a 2′,5′-oligoadenylate (2-5A)-dependent ribonuclease L (RNase L) following virus-infection of mammalian cells. RNase L degrades both viral and cellular RNAs and restricts virus-proliferation. We have studied organization of RNase L gene in genomic DNA from the mouse liver by Southern blot analysis. Several BamHI, BglII, EcoRI, HincII, HindIII, NcoI, PstI, SacI, and XbaI restriction fragments hybridized to ³²P-labeled mouse RNase L cDNA and the 5′-proximal exon probes. Mouse RNase L gene exists as a single copy (>16 kb DNA) gene. A 5 kb HindIII and a 2.5 kb EcoRI DNA were detected as 5′-upstream DNA of the gene which may contain mouse RNase L promoter. Our results will help studying mouse RNase L gene promoter in further details.     

Characterization of small plasmids from Staphylococcus aureus.

Small molecular weight plasmids from Staphylococcus aureus were characterized with respect to size, restriction enzyme cleavage pattern and transforming capacity. The plasmids pS194 and pC194 which encode streptomycin and chloramphenicol resistance respectively contained 3.0 and 2.0 megadaltons of DNA as determined by zonal rate centrifugation and electron-microscopy. Both plasmids transformed S. aureus with high efficiency. Plasmid pC194 contained only one cleavage site for endonuclease HindIII and pS194 contained single cleavage sites for HindIII and EcoRI. A natural recombinant between these two plasmids, pSC194, shared the high transforming capacity of the parental plasmids and contained one EcoRI site And two HindIII sites. pSC194 DNA also transformed B. subtilis with high efficiency. The recombinant plasmid pSC194 may be used as an EcoRI vector for construction and propagation of hybrid DNA in S. aureus as shown in the following paper (Löfdahl et al., 1978).     

Construction and properties of recombinant plasmids containing the rII genes of bacteriophage T4.

Summary The EcoRI digestion products of phage T4 DNA have been examined using a phage DNA transformation assay. A 2.6x10⁶ Dalton fragment was found to contain the rII genes. This fragment was purified and then treated with HindIII endonuclease. The cleavage products were ligated to the vector plasmid pBR313 and viable recombinant plasmids recovered. A genetic assay was employed to demonstrate that the recombinants contained T4 DNA and to localize on the phage genetic map the EcoRI and HindIII sites cleaved during the construction of the plasmids. Preliminary characterization suggests that a fragment covering the beginning of the rIIA gene possibly contains a promotor which is active in uninfected cells.Abbreviations used Ap ampicillin - Tc tetracycline - Mdal 10⁶ Daltons - bp base pairs     

Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6

Summary DNA fragments generated by the EcoRI or HindIII endonucleases from the low copy number antibiotic resistance plasmids R6 and R6-5 were separately cloned using the high copy number ColEl or pML21 plasmid vectors and the insertional inactivation procedure. The hybrid plasmids that were obtained were used to determine the location of the EcoRI and HindIII cleavage sites on the parent plasmid genomes by means of electron microscope heteroduplex analysis and agarose gel electrophoresis. Ultracentrifugation of the cloned fragments in caesium chloride gradients localized the high buoyant density regions of R6-5 to fragments that carry the genes for resistance to streptomycin-spectinomycin, sulfonamide, and mercury and a low buoyant density region to fragments that carry the tetracycline resistance determinant. Functional analysis of hybrid plasmids localized a number of plasmid properties such as resistances to antibiotics and mercury and several replication functions to specific regions of the R6-5 genome. Precise localisation of the genes for resistance to chloramphenicol, kanamycin, fusidic acid and tetracycline was possible due to the presence of identified restriction endonuclease cleavage sites within these determinants.Only one region competent for autonomous replication was identified on the R6-5 plasmid genome and this was localized to EcoRI fragment 2 and HindIII fragment 1. However, two additional regions of replication activity designated RepB and RepC, themselves incapable of autonomous replication but capable of supporting replication of a linked ColE1 plasmid in polA^– bacteria, were also identified.     

Isolation of plasmids present in thermophilic strains from hot springs in Jordan

The plasmid profile of two thermophilic bacterial strains isolated from recreation thermal springs in Jordan has been investigated. These strains are Streptococcus thermophilus and Bacillus sp1, which have been isolated from Zerka – Maeen and Himma hot springs respectively. Supercoiled and circular plasmid forms were detected, explaining the effect of DNA conformation on the mobility of the plasmid in the agarose gel electrophoresis. Two plasmids have been isolated and characterized by restriction endonucleases to facilitate their use as cloning vectors in thermophilic strains. The sizes of the plasmids were approximately 3 kb (from Streptococcus thermophilus) and 7 kb (from Bacillus spl). These plasmids were then digested with three different restriction enzymes (EcoRI, Bam HI, and HindIII), one of which was found to possess a single site for both plasmids, this enzyme is EcoRI.     

Location of Homologous DNA Sequences Interspersed at Five Regions in the Baculovirus AcMNPV Genome

An examination of Autographa californica nuclear polyhedrosis virus DNA revealed the presence of five interspersed regions, rich in EcoRI restriction sites, which shared homologous sequences. These homologous regions (hr), designated hr₁ to hr₅, occur at or near the following EcoRI fragment junctions: hr₁EcoRI-B—EcoRI-I (0.0 map units); hr₂, EcoRI-A—EcoRI-J (19.8 map units); hr₃, EcoRI-C—EcoRI-G (52.9 map units); hr₄, EcoRI-Q—EcoRI-L (69.8 map units); and hr₅, EcoRI-S—EcoRI-X (88.0 map units). Four of these regions were identified, by cross-blot hybridization of HindIII-restricted A. californica nuclear polyhedrosis virus DNA, to be within the HindIII-A/B, -F, -L, and -Q fragments. The location of these regions and the identification of a fifth homologous region were confirmed, and their characterization was facilitated, by using two plasmids with HindIII-L or -Q fragment insertions, which contained the homologous regions hr₂ and hr₅, respectively. The sizes of the homologous regions were about 800 base pairs for hr₂, 500 base pairs for hr₅, and less than 500 base pairs for hr₁, hr₃, and hr₄. A set of small EcoRI fragments (EcoRI minifragments) which ranged in size from 225 to 73 base pairs were detected in A. californica nuclear polyhedrosis virus DNA and HindIII-L and -Q fragments by polyacrylamide gel analysis. Some of the minifragments in viral DNA were present in extramolar amounts and corresponded in size to some of the minifragments present in HindIII-L and -Q. Clones of some of the EcoRI minifragments were used as probes in hybridizations to digests of viral DNA and of HindIII-L and -Q. The hybridization data, obtained under various levels of stringency, suggested that there was a degree of mismatching between the sequences which were responsible for the homology.     

A low-copy-number Sorghum DNA sequence that detects hypervariable EcoRV fragments

A sorghum genomic DNA clone that hybridized on Southern blots in simple but different patterns to fragments produced by digestion of DNA from the parents of an F₂ mapping population was hybridized to EcoRV-digested DNA from 53 accessions. Forty-six different fragment patterns were observed, each comprised of from one to ten bands. Much less variability was detected in EcoRI than EcoRV digests of a selected subset of the accessions. Base-sequence analysis of the clone did not reveal a functional identity for the sequence and the clone does not contain repeated sequences often associated with hypervariable loci. Clones such as this will be especially useful in evaluating germplasm diversity and in identifying the potential parentage of hybrids.     

Subunit structure of chromatin and the organization of eukaryotic highly repetitive DNA: indications of a phase relation between restriction sites and chromatin subunits in African green monkey and calf nuclei.

The periodicities of the restriction enzyme cleavage sites in highly repetitive DNAs of six mammalian species (monkey, mouse, sheep, human, calf and rat) appear related to the length of DNA contained in the nucleosome subunit of chromatin. We suggest that the nucleosome structure is an essential element in the generation and evolution of repeated DNA sequences in mammals (Brown et al., 1978; Maio et al., 1977). The possibility of a phase relation between DNA repeat sequences and associated nucleosome proteins is consistent with this hypothesis and has been tested by restriction enzyme and micrococcal nuclease digestions of repetitive DNA sequences in isolated, intact nuclei.Sites for four different restriction enzyme activities, EcoRI, EcoRI¹, HindIII and HaeIII have been mapped within the repeat unit of component α DNA, a highly repetitive DNA fraction of the African green monkey. The periodicity of cleavage sites for each of the enzymes (176 ± 4 nucleotide base-pairs) corresponds closely to the periodicity (about 185 nucleotide base-pairs) of the sites attacked in the initial stages of micrococcal nuclease digestion of nuclear chromatin. In intact monkey nuclei, EcoRI-RI¹ sites are accessible to restriction enzyme cleavage; the HindIII and HaeIII sites are not. The results suggest (1) that, in component α chromatin, the EcoRI-RI¹ sites are found at the interstices of adjacent nucleosomes and (2) the HindIII and HaeIII sites are protected from cleavage by their location on the protein core of the nucleosome. This interpretation was confirmed by experiments in which DNA segments of mononucleosomes and nucleosome cores released from CV-1 nuclei by micrococcal nuclease were subsequently treated with EcoRI, EcoRI¹ and HindIII. A major secondary segment of component α, about 140 nucleotide base-pairs in length, was released only by treatment with HindIII, in keeping with the location of the HindIII sites in the restriction map and their resistance to cleavage in intact nuclei.EcoRI reduces calf satellite I DNA to a segment of about 1408 nucleotide basepairs. In contrast, restriction of calf satellite I DNA with EcoRI¹ produces six prominent segments ranging in size from 176 to 1408 nucleotide base-pairs. Treatment of isolated calf nuclei with either EcoRI or EcoRI¹ did not produce segments shorter than 1408 base-pairs, indicating that while canonical EcoRI sites are accessible to attack, the irregularly spaced EcoRI¹ sites are specifically blocked. The results are consistent with a phase relation between the repeat sequence of calf satellite I DNA and an octameric array of nucleosomes.     

Family and population studies on the human pepsinogen A multigene family

Summary Human pepsinogen A (PGA) displays highly polymorphic isozymogen patterns after polyacrylamide gel electrophoresis and activity staining. The patterns differ with respect to the presence and the relative intensity of the individual fractions. Family studies strongly suggest that these isozymogen patterns are encoded by allelic haplotypes, encompassing different numbers and types of PGA genes. In this paper, we confirm the essential features of this multigene model. We establish the relationship between the haplotypes and the corresponding isozymogen patterns by determination of the PGA polymorphism at both the DNA and the protein level in 117 Dutch individuals, 60 of whom were unrelated. The combination of HindIII and EcoRI restriction fragment length polymorphisms (RFLPs) has enabled us to define different haplotypes, which are shown to segregate within families. Most genes are characterized by their specific EcoRI fragments. The HindIII RFLP is in strong linkage disequilibrium with PGA genes showing strong expression of the relevant isozymogen. Although a general picture of the relationship between genotypes and phenotypes is emerging, there are exceptions, suggesting that rare haplotypes evolve by unique crossover events.     

Chip ligating human genomic DNA serves as storage material and template for polymerase chain reaction

A chip was developed to store DNA for medical research. The optional restriction site fixed on the chip can randomly ligate with whole human genomic DNA treated by the corresponding restriction enzyme. PCR can then use the chip as template DNA. Moreover, a chip fixing two restriction sites (e.g. EcoRI and HindIII) showed the amplification by PCR for any location of genomic DNA. Repetitive PCRs have confirmed that a DNA chip can be stored by at –4 °C for 2 years.     

Mitochondrial DNA from Podospora anserina

Summary EcoRI fragments of the 94 kilobase mitochondrial DNA (mtDNA) from young, wild type Podospora anserina were cloned into the EcoRI site of the E. coli plasmid vector pBR325. A complete EcoRI clone bank was developed, containing all 16 of the EcoRI fragments from the native mtDNA. Restriction endonuclease maps for the enzymes SalI, XhoI, BamHI, EcoRI, BglII, and HaeIII were constructed from the analysis of single, double, and triple restriction digests of cloned and native mtDNA. In constructing the maps data were refined by extensive Southern analysis of the native genome hybridized to cloned DNA probes. Restriction maps were analyzed and permitted us to locate the origin of mtDNA derived from senescent cultures.Both the large and small rRNA genes were then localized on these restriction maps using Southern and Northern blot analysis. We have shown the large rRNA locus to lie within a 10.8 kb region of EcoRI fragments E5 and E7, and the small rRNA locus to lie on a 5 kb subfragment of EcoRI fragment E1. The limit of separation between these two loci was determined to be between 6 and 9 kb.Surprisingly, when electrophoresed in agarose-CH₃HgOH gels, the large rRNA was found to be 3.8 kb long, 500 bases longer than that from the very closely related Neurospora crassa, making it the largest rRNA yet described.     

Plasmid vectors derived from phage Mu allow direct selection of transformants containing cloned HindIII and PstI fragments

Summary The vector plasmids pKN001 and pKN80 both contain the EcoRI.C fragment of E.coli phage Mu DNA which codes for a killing function that is efficiently expressed upon transformation into Mu-sensitive bacteria. By in vitro insertion of HindIII fragments at the single HindIII site of pKN80 or of PstI fragments at the single PstI site of pKN001 the killing function is inactivated. The resulting plasmids have a selective advantage over the religated vector when transformed into Mu-sensitive bacteria. More than 90% of the transformants contain hybrid plasmids. These results show the usefulness of Mu DNA containing plasmids pKN001 and pKN80 as vectors that allow the direct selection for recombinant plasmids.     

Localization of the active gene of aldolase on chromosome 16, and two aldolase A pseudogenes on chromosomes 3 and 10

Summary Southern blot analysis of human genomic DNA hybridized with a coding region aldolase A cDNA probe (600 bases) revealed four restriction fragments with EcoRI restriction enzyme: 7.8 kb, 13 kb, 17 kb and >30 kb. By human-hamster hybrid analysis (Southern technique) the principal fragments, 7.8 kb, 13 kb, >30 kb, were localized to chromosomes 10, 16 and 3 respectively. The 17-kb fragment was very weak in intensity; it co-segregated with the >30-kb fragment and is probably localized on chromosome 3 with the >30-kb fragment. Analysis of a second aldolase A labelled probe protected against S1 nuclease digestion by RNAs from different hybrid cells, indicated the presence of aldolase A mRNAs in hybrid cells containing only chromosome 16. Under the stringency conditions used, the EcoRI sequences detected by the coding region aldolase A cDNA probe did not correspond to aldolase B or C. The 7.8-kb and >30-kb EcoRI sequences, localized respectively on chromosomes 10 and 3, correspond to aldolase A pseudogenes, the 13-kb EcoRI sequence localized on chromosome 16 corresponds to the aldolase active gene. The fact that the aldolase A gene and pseudogenes are located on three different chromosomes supports the hypothesis that the pseudogenes originated from aldolase A mRNAs, copied into DNA and integrated in unrelated chromosomal loci.     

Chloroplast ribosomal RNA genes in Euglena gracilis exist as three clustered tandem repeats

Chloroplast ribosomal DNA from Euglena gracilis was partially purified, digested with restriction endonucleases BamHI or EcoRI and cloned into bacterial plasmids. Plasmids containing the ribosomal DNA were identified by their ability to hybridize to chloroplast ribosomal RNA and were physically mapped using restriction endonucleases BamHI, EcoRI, HindIII and HpaI. The nucleotide sequences coding for the 16S and the 23S chloroplast ribosomal RNAs were located on these plasmids by hybridizing the individual RNAs to denatured restriction endonuclease DNA fragments immobilized on nitrocellulose filters. Restriction endonuclease fragments from chloroplast DNA were analyzed in a similar fashion. These data permitted the localization on a BamHI map of the chloroplast DNA three tandemly arranged chloroplast ribosomal RNA genes. Each ribosomal RNA gene consisted of a 4.6 kilobase pair region coding for the 16S and 23S ribosomal RNAs and a 0.8 kilobase pair spacer region. The chloroplast ribosomal DNA represented 12% of the chloroplast DNA and is G + C rich.     

Chloroplast DNA restriction fragment length polymorphism in Sequoia sempervirens D. Don Endl., Pseudotsuga menziesii (Mirb.) Franco,Calocedrus decurrens (Torr.), and Pinus taeda L.

Summary The extent and type of chloroplast DNA restriction fragment length polymorphism was determined among individual tree samples of coast redwood, Douglas fir, incense-cedar, and loblolly pine. A total of 107 trees was surveyed for three restriction enzymes (BamHI, EcoRI, HindIII) and six chloroplast DNA probes from petunia (P3, P4, P6, P8, P10, S8). The probes comprise 64% of the petunia chloroplast genome. Polymorphisms were detected in all species but loblolly pine. Coast redwood and incense-cedar had a small number of rare variants, whereas Douglas fir had one highly polymorphic region of insertions/deletions in sequences revealed by the P6 probe from petunia. The mutation hotspot is currently being studied by DNA sequence analysis.     

Restriction endonuclease analyses of the incompatibility group P-1 plasmids RK2, RP1, RP4, R68, and R68.45

The P-group plasmids RP1, RP4, RK2, R68 and R68.45 were analyzed by the following restriction endonucleases:BamHI,BglII,EcoRI,HindIII,PstI,PvuII,SalI, andSmaI. No differences between RP1, RP4, and RK2 were found, and the plasmid R68.45 was found to contain a direct duplication of an existing DNA sequence in R68. Our map of RK2 differs from the published map of RK2 in the corresponding region of the R68 map that is duplicated in R68.45.     

Restriction Fragment Length Polymorphism Analyses of the Plasmid DNAs in Strains of Xanthomonas campestris pv. vesicatoria from Different Geographic Areas

Restriction fragment length polymorphisms (RFLPs) of plasmid DNAs in Xanthomonas campestris pv. vesicatoria were analysed using 77 strains from the United states, Argentina, Australia, Taiwan, and Korea. One or more plasmids were detected in all tested strains, irrespective of geographic origin, host plant from which isolated, or chemical resistance. All Korean strains contained a few plasmids of similar high molecular weight, whereas some small plasmids occured only in strains from the United States, Argentina, and Taiwan. After digesting total plasmid DNAs with each of four restriction endonucleases, 18 fragments with sizes from about 1 to 23 kb were visualized. Seventy-seven strains of diverse geographic origins, with different levels of resistance to streptomycin and copper, were classified into the 14 RFLP groups based on the restriction endonuclease digestion patterns of their plasmid DNAs. Strains belonging to each group shared DNA fragments of identical size, suggesting the possible presence of similar plasmids in these strains. A 5.8-kb EcoRI plasmid DNA probe prepared from the United States strain 81-23 hybridized to EcoRI plasmid digests from all tested strains. Other plasmid DNA fragments of the strain81-2,3 used as probes had no homology to plasmid DNA fragments from several strains around the world. The variation in hybridization profiles of plasmid DNA was very similar to the results obtained by RFLP analysis of plasmid DNA digested by four restriction enzymes. Most of the Korean strains tested were highly sensitive to streptomycin and copper, whereas most strains from other geographic areas showed a high level of resistance to one or two of the chemicals. Cluster analysis of genetic distance between the strains based on the data obtained generated the dendrograms that separated all Korean strains from the other strains, suggesting that plasmid DNA of the Korean strains may be genetically very different from those of the others.     

Physical characterization of the plasmid pON5300

Summary The antibiotic resistance plasmid Rldrd19Km-which has spontaneously lost its kanamycin resistance marker, and its derivative pON5300, were analysed using the restriction endonucleases SalI, BamHI, HindIII and EcoRI. The fragment patterns were compared with that of the Rldrd19 and the fragments responsible for kanamycin resistance were found to be missing in Rldrd19Km ^– and pON5300. In these plasmids a 7 Mg/mol EcoRI fragment was observed instead of the D (6.3 Mg/mol) fragment of Rldrd19. Further a new 6 Mg/mol EcoRI restriction fragment was observed in pON5300. Using double digestions it was shown that the new fragment does not carry restriction sites for HindIII, BamHI and SalI endonucleases. The non-homology of the analysed plasmid was proved electron microscopically by heteroduplex techniques. The possibility of amplification in the regulatory region for the expression of R-determinants in pON5300 is discussed.