EcoRI Restriction Fragment Research Articles

Conservation genetics of the koala (Phascolarctos cinereus) II. Limited variability in minisatellite DNA sequences

We have examined variability in TaqI and EcoRI restriction fragment sizes of DNA from the koala (Phascolarctos cinereus) using six HVR (hypervariable region) probes which reveal complex, individual-specific restriction patterns in humans and other species. Frequency of band-sharing among unrelated koalas was extremely high. This result is likely to be a consequence of the history of near-extinction and artificial recolonization of the populations we have studied, rather than a general marsupial or koala-wide phenomenon.

Microclones derived from the mouse chromosome 7 D-E bands map within the proximal region of the c14 CoS deletion in albino mutant mice

A group of radiation-induced perinatal-lethal deletions that include the albino ( c) locus on mouse chromosome 7 causes failure of expression of various hepatocyte-specific genes when homozygous ( S. Gluecksohn-Waelsch, 1979, Cell 16: 225–237). The transcription of such genes could be controlled in trans by a regulatory gene(s) located within the proximal region of the c 14 CoS deletion. To identify this potential regulatory gene, a microclone library was established from microdissected D and E bands of chromosome 7. Three nonoverlapping microclones (E305, E336B, and E453B) hybridizing with wildtype but not with c 14CoS c 14CoS DNA were isolated. E336B represents a single-copy DNA fragment, whereas E305 and E453B hybridized with 3 and 10 EcoRI DNA restriction fragments, respectively. All fragments map exclusively within the deletion. The microclones hybridized to DNA of viable c 6H c 14CoS deletion heterozygotes but not to DNA of homozygotes for the lethal mutation c 10 R75 M , which belongs to the same complementation group as c 14 CoS . DNA of viable homozygous mutant c 62 DSD , which carries a deletion breakpoint proximal to that of c 6 H , hybridized only with E453B. This microclone identified 6 EcoRI restriction fragments in c 62DSD c 62DSD DNA. The results demonstrate that of the isolated microclones, E453B identifies a locus (D7RT453B) that maps closest to the hsdr-1 (hepatocyte-specific developmental regulation) locus ( S. Gluecksohn-Waelsch, 1989, Mouse Newslett. 83: 149), which maps between the proximal breakpoints of deletions c 10 R75 M and c 62 DSD .

Identification and molecular characterization of the Alcaligenes eutrophus H16 aco operon genes involved in acetoin catabolism

Acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) and the fast-migrating protein (FMP) were purified to homogeneity from crude extracts of acetoin-grown cells of Alcaligenes eutrophus. Ao:DCPIP OR consisted of alpha and beta subunits (Mrs, 35,500 and 36,000, respectively), and a tetrameric alpha 2 beta 2 structure was most likely for the native protein. The molecular weight of FMP subunits was 39,000. The N-terminal amino acid sequences of the three proteins were determined, and oligonucleotides were synthesized on the basis of the codon usage of A. eutrophus. With these, the structural genes for the alpha and beta subunits of Ao:DCPIP OR and FMP, which were referred to as acoA, acoB, and acoC, respectively, were localized on one single EcoRI restriction fragment which has been cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The nucleotide sequences of a 5.3-kbp region of this fragment and one adjacent fragment were determined, and the structural genes for acoA (1,002 bp), acoB (1,017 bp), and acoC (1,125 bp) were identified. Together with the gene acoX, whose function is still unknown and which is represented by a 1,080-bp open reading frame, these genes are probably organized in one single operon (acoXABC). The transcription start site was identified 27 bp upstream of acoX; this site was preceded by a region which exhibited complete homology to the enterobacterial sigma 54-dependent promoter consensus sequence. The amino acid sequences deduced from acoA and acoB for the alpha subunit (Mr, 35,243) and the beta subunit (Mr, 35,788) exhibited significant homologies to the primary structures of the dehydrogenase components of various 2-oxo acid dehydrogenase complexes, whereas those deduced from acoC for FMP (Mr, 38,941) revealed homology to the dihydrolipoamide acetyltransferase of Escherichia coli. The occurrence of a new enzyme type for the degradation of acetoin is discussed.

Serial Alu sequence transposition interrupting a human B creatine kinase pseudogene

We have isolated, sequenced, and characterized a single-copy B creatine kinase pseudogene. The chromosomal assignment of this gene is 16p13 and a unique sequence probe from this locus detects EcoRI restriction fragment length polymorphisms of 7.8 and 5.4 kb. In 26 unrelated individuals, the frequencies for the 7.8- and 5.4-kb B creatine kinase pseudogene alleles were calculated to be 17.3 and 82.7%, respectively. The B creatine kinase pseudogene is interrupted by a 904-bp DNA insertion composed of three Alu repeat sequences in tandem flanked by an 18-bp direct repeat, derived from the pseudogene sequence. Nucleotide sequence analysis of the Alu elements suggests that the Alu sequences were incorporated into this locus in three separate integration events. Several complex clustered Alu repeat sequences without defined integration borders have been previously identified at different genomic loci. This is the first evidence that complex tandem Alu elements can integrate in an apparently serial manner in the human genome and supports the contention that Alu repeats integrate nonrandomly into the human genome.

Fragile X Genotype Characterized by an Unstable Region of DNA

DNA sequences have been located at the fragile X site by in situ hybridization and by the mapping of breakpoints in two somatic cell hybrids that were constructed to break at the fragile site. These hybrids were found to have breakpoints in a common 5-kilobase Eco RI restriction fragment. When this fragment was used as a probe on the chromosomal DNA of normal and fragile X genotype individuals, alterations in the mobility of the sequences detected by the probe were found only in fragile X genotype DNA. These sequences were of an increased size in all fragile X individuals and varied within families, indicating that the region was unstable. This probe provides a means with which to analyze fragile X pedigrees and is a diagnostic reagent for the fragile X genotype.

Characterization of PDR4, a Saccharomyces cerevisiae gene that confers pleiotropic drug resistance in high-copy number

PDR4 is a gene that confers pleiotropic drug resistance (pdr) to the yeast Saccharomyces cerevisiae when present in high copy number [Leppert et al., Genetics 125 (1990) 13–20]. Transposon insertion mutations had identified the active region of the gene as a 3.7-kb SalI- EcoRI restriction fragment of the 8-kb cloned fragment. We have confirmed this by showing that this fragment is sufficient to confer pdr, and have sequenced its entire 3761 bp. It contains a single complete open reading frame (ORF) extending from nucleotide (nt) position 1631–3580, coding for a protein of 650 amino acids (aa). A 2.7-kb fragment containing this ORF is also sufficient to confer pdr. The aa sequence contains no recognizable homologies or consensus sequences, so it is a novel protein of unknown function. It is apparently soluble, since no transmembrane-type sequences were predicted. A second, partial ORF was also found, on the opposite strand, extending from nt position 774 to past the SalI site, which is apparently unrelated to pdr.

Long-range restriction mapping and linkage analysis of the Prader-Willi chromosome region ( PWCR)

In an attempt to elucidate the relationship between genetic alterations at chromosomal bands 15q11.2–12 and the Prader-Willi syndrome (PWS), we have constructed a long-range restriction map of this region using a combination of pulsed-field gel techniques and the infrequently cutting restriction enzymes NotI, MluI, SalI, SfiI, NruI, SacII, and BssHII. Four previously reported probes mapping to 15q11.2–12 and known to be deleted in PWS patients were used to construct the physical map of this region. The loci recognized by these four probes have been localized to a 2600-kb partial SalI restriction fragment and a 3200-kb partial EcoRI restriction fragment. Linkage studies were performed on nine families to estimate the recombination rates between these loci. The calculated lod scores did not indicate significant linkage between any of the four loci. The contrast between the physical distance and the observed recombination frequency suggests that these four loci are located in a recombinational “hot spot”.

Repetitive Sequences with Homology toBradyrhizobium japonicumDNA and the T-DNA ofAgrobacterium rhizogenesAre Closely Linked tonodABCofRhizobium frediiUSDA257

We have detected strong homology between a 9.2-kb EcoRI restriction fragment from Rhizobium fredii USDA257 that contains nodABC and eight additional EcoRI fragments in DNA digests from this organism. A series of repetitive sequences responsible for this hybridization lies within a 0.95-kb HindIII/SalI subfragment about 1-kb upstream of nodA. This subfragment also hybridizes to multiple restriction fragments from nine other strains of R. fredii, but only one is common to all strains. The 0.95-kb subfragment does not hybridize to genomic DNA from 17 other strains of fast-growing rhizobia, but there is weak homology to two fragments from Rhizobium sp. strain NGR234. We sequenced 2,432 base pairs (bp) of the region encompassing the repetitive sequences. It contains 65 separate 8- to 11-bp inverted and direct repeats, as well as two large open reading frames (ORFs) that overlap on opposite strands. ORF1 reads in the same direction as nodABC, contains 1,071 bp, and encodes a 40.6-kD protein. It has 74% sequence homology to an ORF within the T-DNA of Agrobacterium rhizogenes and similar homology to a series of repetitive sequences from Bradyrhizobium japonicum. ORF2 (981 bp) reads in the opposite direction, encodes a 34.7-kD protein, and has partial identity with a second ORF from A. rhizogenes. We could detect no poly(A)+ nodule transcripts with homology to ORF1 and ORF2. The eight sets of repetitive sequences found in other EcoRI fragments of the genome were cloned from USDA257 on separate cosmids. Some of these cosmids appear to overlap, and two have fragments with homology to nifKDH.

Diverse forms of stress results in changes in cellular levels of osteonectin/SPARC without altering mRNA levels in osteoligament cells

The osteonectin/SPARC gene has been shown to possess motifs for a heat shock element and metal responsiveness. Also, the expression of the protein has been associated with culture stress in endothelial cells. In the present study, osteoligament (OL) cells derived from the patellar ligament were subjected to diverse forms of stress that included (a) exposure to sodium arsenite, (b) heat shock, (c) cadmium ion, and (d) the amino acid analog, AZC. Osteonectin/SPARC levels in OL cells were determined by Western blot analyses, and immunoprecipitation using antiosteonectin antibodies. Expression of osteonectin/SPARC mRNA was determined by Northern analysis using a 1.5 kb EcoRI restriction fragment of bovine osteonectin cDNA. These studies reveal that osteonectin/SPARC is produced following diverse forms of stress, however, the levels are lower than observed in unchallenged OL cells. In all instances, the mRNA levels were comparable to control cells. These studies indicate that expression of osteonectin/SPARC mRNA is tightly controlled in OL cells and that the protein may be regulated at the level of protein translation.

Identification of mRNA that encodes an alternative form of H-CAM(CD44) in lymphoid and nonlymphoid tissues

The cluster of differentiation 44 (CD44), hereafter referred to as H-CAM(CD44), represents a novel class of polymorphic (Mr 80,000-215,000) cell adhesion molecules that are involved in cell-cell and cell-matrix adhesion events in a variety of organ systems. We report the detection of distinct mRNAs, in both hematopoietic and nonhematopoietic human cell lines, that encode H-CAM(CD44) with different cytoplasmic domains. Genomic Southern blot analyses indicate that the exons encoding these two cytoplasmic domains are located on the same approximately 16 kilobase (kb) Eco RI restriction fragment. Restriction endonuclease and Southern blot analyses performed on polymerase chain reaction (PCR) amplification copies of these mRNAs confirm that their sequences correspond with previously reported cDNA sequences. A consensus splice donor site which is conserved in human, baboon, and mouse mRNAs that encode a molecule with an elongated cytoplasmic domain (H-CAM-L) is utilized to generate a distinct but low-abundance mRNA species that encodes H-CAM(CD44) with a truncated cytoplasmic domain of only three amino acids (H-CAM-S). Estimations of the relative abundance of these mRNA species in B-lymphoblastoid cells using the PCR amplification technique exhibit average H-CAM-L/H-CAM-S ratios ranging between 100 and 200. Therefore, H-CAM (CD44)-mediated adhesive events may be regulated through a differential capacity of H-CAM-L and H-CAM-S to interact with the cytoskeleton and to participate in intracellular signaling events.

Assessment of the degree and the type of restriction fragment length polymorphism in barley (Hordeum vulgare)

In order to determine the extent of polymorphism in barley (Hordeum vulgare), DNA from 48 varieties was analyzed with 23 genomic, single-copy probes, distributed across all seven chromosomes. Upon hybridization to wheat-barley addition lines, the probes showed different degrees of homology compared to the wheat genome. Polymorphisms were detected in the barley genome at a frequency of 43% after digestion with EcoRI, BamHI, and HindIII. Subgroups of spring and winter barley and of two- and six-rowed types showed less diversity which, in most cases, was due to shifts in allelic frequencies. One probe (MWG1H504) hybridized to an EcoRI restriction fragment exclusively observed in winter barley. A comparison of six different restriction enzymes revealed clear differences with regard to their efficiency in detecting polymorphisms. The respective frequencies were between 13% (HindIII) and 37% (EcoRV). A significant correlation between the efficiency of a restriction enzyme and the mean fragment size detected by the different probes identified insertion/deletion events as the major factor causing polymorphism in barley.

Cloning and sequencing of the celA gene encoding endoglucanase A of Butyrivibrio fibrisolvens strain A46

Genomic DNA from Butyrivibrio fibrisolvens strain A46 was digested with EcoRI and ligated into lambda gt11. Two recombinant phages isolated from the gene bank hydrolysed carboxymethylcellulose and were shown to contain the same 2.3 kb EcoRI restriction fragment, which was cloned into pUC12 to generate pBA46. Escherichia coli JM83 harbouring pBA46 expressed an endoglucanase (EGA) which hydrolysed a range of other substrates including barley beta-glucan, Avicel, filter paper and p-nitrophenyl beta-D-cellobioside. Nucleotide sequencing of the B. fibrisolvens strain A46 DNA cloned in pBA46 revealed a single open reading frame (ORF) of 1296 bp, encoding a protein of 48,863 Da. Confirmation that the ORF coded for EGA was obtained by comparing the N-terminal sequence of the purified endoglucanase with that deduced from the nucleotide sequence. EGA contains a typical prokaryotic signal peptide at its N-terminus and shows some homology with the Bacillus family of cellulases. The enzyme does not contain distinct functional domains, which are prevalent in cellulases from Pseudomonas fluorescens subsp. cellulosa and Cellulomonas fimi.

Developmental cycle-specific host-free RNA synthesis in Chlamydia spp

The incorporation of radiolabeled GTP into RNA in host-free Chlamydia trachomatis serovar L2 organisms was investigated. The incorporation was partially inhibited by rifampin and dactinomycin and hydrolyzed by RNase. RNA made by host-free chlamydiae consisted mainly of species of fewer than 800 bases in size, although 16S and 23S species were noted by agarose-gel electrophoresis. The hybridization of radiolabeled host-free RNA to restriction fragments of the gene encoding the major outer membrane protein was analyzed; all regions of the gene were transcribed. The relative intensity of hybridization of host-free RNA made by chlamydiae isolated during the middle and late stages of the developmental cycle to the DNA of clones encoding gene products known to be made at these times in vivo indicated that the temporal patterns of host-free and in vivo transcription were similar. Radiolabeled RNA from 1- and 24-h host-free Chlamydia psittaci 6BC organisms hybridized to many of the same EcoRI and BamHI restriction fragments of C. psittaci genomic DNA, although some differences could be noted. When these RNAs were used to screen a partial C. psittaci genomic library in lambda gt11, plaques were identified that reacted mainly either with 1-h RNA or with 24-h RNA. Because RNA synthesized by host-free chlamydiae appears to be developmental cycle stage specific, transcripts made by host-free chlamydiae may be convenient probes that can be used to clone developmental stage-specific chlamydial genes.

Integration of an unstable plasmid into the chromosome of Lactobacillus plantarum

A 2.3 kb Eco RI restriction fragment of Lactobacillus plantarum genomic DNA was cloned into pSA3 to generate pJRI. Both pSA3 and pJR1 were transformed into L. plantarum . Growth of the transformants in the absence of the selection pressure, erythromycin (both plasmids confer Emr), led to a stable sub-population of EMr bacteria in which pSA3 and pJR1 had integrated into the L. plantarum genome. Amplification of the chromosomally located plasmids was observed when the organism was grown in the presence of erythromycin. The integrated plasmid sequences were stably maintained in the absence of selection pressure, and did not affect the growth rate of the lactic acid bacterium in rich media.

Integration of an unstable plasmid into the chromosome of Lactobacillus plantarum

A 2.3 kb EcoRI restriction fragment of Lactobacillus plantarum genomic DNA was cloned into pSA3 to generate pJRI. Both pSA3 and pJR1 were transformed into L. plantarum. Growth of the transformants in the absence of the selection pressure, erythromycin (both plasmids confer Em r), led to a stable sub-population of EM r bacteria in which pSA3 and pJR1 had integrated into the L. plantarum genome. Amplification of the chromosomally located plasmids was observed when the organism was grown in the presence of erythromycin. The integrated plasmid sequences were stably maintained in the absence of selection pressure, and did not affect the growth rate of the lactic acid bacterium in rich media.

Candida albicans in Patients with the Acquired Immunodeficiency Syndrome: Absence of a Novel or Hypervirulent Strain

To determine whether Candida albicans in patients with AIDS represents a unique strain, C. albicans isolated from 24 patients with AIDS was compared with Candida isolated from 23 healthy adults. Resistance to 5-fluorocytosine, synthesis of amino acids and nucleotides, sugar use, and enzyme activity patterns were similar among isolates from the two groups. Molecular analysis revealed similar banding patterns of EcoRI restriction fragments of DNA between 2.5-3 and 6-7 kb. In addition, the frequency of a dimorphic 3.7-versus 4.2-kb band, identified by agarose gel electrophoresis and by probing Southern transfers of EcoRI digests with a cloned fragment of C. albicans DNA encoding 25S ribosomal RNA, was not significantly different between the AIDS-derived and control C. albicans. C. albicans isolated at different time points in the course of disease and from different sites in individual patients showed identical DNA fingerprints. The similarity in isolates of C. albicans that cause disease in AIDS patients and those present in healthy subjects suggests that the candidiasis associated with AIDS is not due to the presence of a unique or particularly virulent strain but is likely the consequence of a defect in host defense mechanisms.

Studies on Cyanidium caldarium Phycobiliprotein Pigment Mutants

Phycobiliprotein biosynthesis was investigated in four strains of the unicellular rhodophyte, Cyandium caldarium, with different pigment phenotypes. All strains were incapable of synthesizing phycobiliproteins when grown in the dark. Western blotting experiments showed that dark-grown cells of the wild-type and mutant GGB synthesized the alpha and beta subunit polypeptides of allophyocyanin and phycocyanin after exposure to light for 24 hours, whereas cells of mutant IIIC and GGBY did not. Similarly, light promoted the appearance of allophycocyanin and phycocyanin mRNAs in the wild-type and GGB but not in IIIC and GGBY. However, Southern blots of restricted genomic DNA from the wild type, IIIC, GGBY, and GGB, all hybridized with heterologous phycobiliprotein gene probes and revealed that all four strains contained identical Pst, EcoRI, and Dral restriction fragments containing allophycocyanin and phycocyanin genes. Cells of the wild type and GGB incubated in the dark with the heme precursor. delta-aminolevulinate, synthesized allophycocyanin and phycocyanin apoproteins providing strong evidence for the role of a tetrapyrrole in regulation of phycobiliprotein gene expression. However, cells of IIIC and GGBY incubated in the dark with delta-aminolevulinate did not contain detectable quantities of allophycocyanin or phycocyanin apoproteins. The possible role of a tetrapyrrole in phycobiliprotein gene expression and basis for the genetic lesion in mutants IIIC and GGBY is discussed.

Differential expression of the c-erbB-2 gene in human small cell and non-small cell lung cancer : Schneider PM, Hung M-C, Chiocca SM et al. Department of Thoracic Surgery, University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030. Cancer Res 1989;49:4968-71

We studied non-small cell lung cancers (NSCLC) from 60 patients for abnormalities in the c-erbB-2 gene. Eleven human lung cancer cell lines, including four derived from small cell lung cancer (SCLC) and seven derived from NSCLC were also examined for altered c-erbB-2 gene expression. Southern blot analysis of paired tumor and normal lung samples demonstrated that amplification of the c-erbB-2 gene is rare in NSCLC (2/60) and not restricted to adenocarcinomas. One patient showed an EcoRI restriction fragment length polymorphism for the c-erbB-2 locus. Four of four SCLC cell lines demonstrated minimal or nondetectable expression of c-erbB-2 mRNA compared to high levels of expression by seven of seven NSCLC lines. The highest expression levels were seen in four of four adenocarcinomas. We conclude that c-erbB-2 expression is different in SCLC compared to NSCLC and high expression of c-erbB-2 is consistently present in lung adenocarcinomas.

Physical characterisation of the replication region of the Streptococcus faecalis plasmid pAMβ1

The complete nucleotide (nt) sequence of a 5.1-κb Eco RI ONA restriction fragment carrying the replication region of the Streptococcus faecalis plasmid pAMβ1 has been determined. Ofthe seven major open reading frames (ORF A-G) identified within this fragment, two (C and E) were shown to be encoding by in vitro transcription/translation assays. Evidence was obtained that synthesis of the polypeptide (M, 57380) encoded by the largest ORF (E) was essential for replication. Deletion analysis indicated that the minimum unit of DNA required for replication resided on a 2.59-κb AccI- HpaI subfragment. ORF C resided outside of this fragment and encompassed an extensive region of directly repeated nt sequence. The encoded polypeptide (Mr, 30471) was therefore composed oflarge tracts of reiterated amino acid sequence (11 × VDP and 35 × TEP tripeptides) which probably caused the observed anomalous electrophoretic mobility of the synthesised protein (equivalent to 61 κDa). Deletion of a 416-bp segment of DNA between unique KpnI and StyI sites caused an increase in copy number, which correlated with the in vitro production of higher levels of 0 RF E polypeptide. Although homology was detected between the sequenced DNA, and the replicon of a closely related streptococcal plasmid (pSM19035),none was evident to any other characterised Gram+ plasmid.

Identification of an abnormal gene for the GPIIIa subunit of the platelet fibrinogen receptor resulting in Glanzmann's thrombasthenia

The platelet fibrinogen receptor, which is composed of glycoproteins IIb (GPIIb) and IIIa (GPIIIa), belongs to a large family of receptors that participate in a multitude of biologically important adhesive interactions. Platelets from most patients with the autosomal recessive bleeding disorder, Glanzmann's thrombasthenia, are deficient in GPIIb and GPIIIa. We have used cDNA probes to analyze the GPIIb and GPIIIa genes in four patients from three kindreds with Glanzmann's thrombasthenia. Southern analysis of their DNA was identical to that observed in normals when probed with a full-length GPIIb cDNA or a 3′ GPIIIa cDNA. However, in one family, a 5′ 2.0 kb GPIIIa cDNA identified abnormal DNA fragments in the father and two affected siblings' genes. A series of restriction digests resulting in small genomic fragments were probed with portions of the 5′ 2.0 kb GPIIIa cDNA and indicated that the abnormal sequences are flanked by normal fragments of the GPIIIa gene. To analyze further the genetic defect in this family, RNA was prepared from their platelets. Northern analysis revealed normal levels of GPIIb mRNA compared to control platelets. We were unable to identify GPIIIa mRNA of any size in the clinically affected family members. We also identified an EcoRI restriction fragment length polymorphism (RFLP) that permitted carrier status determination in the clinically unaffected siblings. These studies indicate that Glanzmann's thrombasthenia can be caused by heterogeneous defects in the GPIIIa gene. Furthermore, we have shown that platelets can be used to characterize normal and abnormal GPIIIa and GPIIb mRNA, and RFLPs may be used to determine the carrier status in some families with Glanzmann's thrombasthenia. The specific gene abnormality in this family appears to represent an example of an insertional mutation resulting in a human disease.