EcoRI Digestion Research Articles

Polyclonal rearrangements of the T‐cell receptor β‐chain in fatal angioimmunoblastic lymphadenopathy

Genomic rearrangement of germline T-cell antigen receptor (TcR) and immunoglobulin (Ig) genes was studied by Southern blot analysis in seven patients with angioimmunoblastic lymphadenopathy (AILD). In three cases clinically suspected of transformation into malignant lymphoma, hybridization with the TcR beta probe showed markedly dimished intensity in the 11.5 kb germline band after Eco RI digestion and normal germline configuration after Hind III and Bam HI digestion, indicating polyclonal T cell rearrangements. A clonal rearrangement of the TcR beta gene was detected in only one case at initial biopsy. No monoclonal rearrangement of Ig genes was observed. These data show that in some cases of AILD disease progression is indicated by polyclonal TcR rearrangements and not by outgrowth of a malignant clone, supporting the concept of AILD as an immunoregulatory disorder.

A tissue-specific change in repetitive DNA in rats.

From a genomic library constructed from an EcoRI digest of Wistar rat brain DNA, we isolated a clone (BL-1) that gave a 0.6-kilobase restriction fragment only in brain (or lens) DNA upon Southern hybridization. The tissue-specific fragment was present in Wistar and other strains (Sprague-Dawley and Donryu) of rats regardless of their sex and age. Sequencing of the clone indicated that it is closely related to a part of the LINE 3 sequence, one of the highly repetitive sequences present throughout mammalian genomes. Polymerase chain reaction using primer sequences in the BL-1 clone indicated that it is derived from an amplified (rear-ranged) sequence, although other explanations are possible. These results suggest that there are tissue-specific changes in DNA primary structure during mammalian developmental processes.

DNA polymorphism among isolates from multiple sites of a patient with chronic herpes simplex virus, type 1 infection.

DNA polymorphisms among independent isolates of herpes simplex virus (HSV) type 1 were studied from a 7-year-old male patient with recurrent infections of the skin and internal organs. In the patient's serum, HSV antibodies could not be detected by complement fixation, enzyme-linked immunosorbent assay (ELISA), or neutralization tests. ELISA tests for the presence of antibodies to human immunodeficiency virus were also negative. One HSV isolate was obtained from mesenteric nodes biopsied in 1983; one from skin in 1984; and three (postmortem) from brain, lungs, and liver in 1985. Restriction enzymes Eco RI, Bgl II, Hind III, Kpn I, and Bam H1 digestion patterns of the five isolates were similar. However, Sal I digests of isolates from skin, mesenteric nodes, lungs, and liver showed variations that were distinct from that of the brain isolate. Although Sal I digests of skin, mesenteric nodes, lungs, and liver isolates share a common variation in lacking F and G, the liver isolate can be further differentiated because of the gain of a restriction site on the H fragment. Thus, the three distinct variants observed were the isolates from brain (variant 1); from skin, mesenteric nodes, and lungs (variant 2); and from liver (variant 3). The fragments involved in variations among these isolates (presence or absence of Sal, G and H) are from the unique short and long regions (invariable regions) of the genome and therefore do not show heterogeneity in size. The extent of variation among these isolates is less than that seen among epidemiologically unrelated strains, suggesting that they originated from a single infecting strain, probably the brain isolate.

Multiplicity of TEM-derived beta-lactamases from Klebsiella pneumoniae strains isolated at the same hospital and relationships between the responsible plasmids

Five plasmid-mediated beta-lactamases conferring high-level resistance to ceftazidime were isolated from Klebsiella pneumoniae strains in the same hospital. These enzymes had isoelectric points ranging from 5.3 to 6.5 (CAZ-1, 5.55; CAZ-2, 6.0; CAZ-3, 5.3; CAZ-6, 6.5; and CAZ-7, 6.3). All isolates and their Escherichia coli transconjugants were highly resistant to amoxicillin (MICs, greater than 4,096 micrograms/ml), piperacillin (64 to 256 micrograms/ml), cephalothin (32 to 256 micrograms/ml), and ceftazidime (32 to 512 micrograms/ml) but remained moderately susceptible to cefotaxime (0.5 to 8 micrograms/ml). Only CAZ-6- and CAZ-7-producing strains were highly resistant to aztreonam (64 to 128 micrograms/ml). All the isolates remained susceptible to moxalactam and imipenem. The reduced activity of piperacillin, cefotaxime, ceftazidime, or aztreonam was restored by 2 micrograms of clavulanate, sulbactam, tazobactam, or brobactam per ml for E. coli producing CAZ-2, CAZ-3, and CAZ-7. Sulbactam had a lower protective effect than other inhibitors for E. coli harboring CAZ-1 and especially CAZ-6. Except for CAZ-1, which was mediated by a 150-kilobase (kb) plasmid (pCFF14), the other ceftazidimases were mediated by plasmids of 85 kb with EcoRI digestion patterns similar to that of pCFF04 encoding CTX-1 beta-lactamase. A TEM probe hybridized with a 19-kb EcoRI fragment of all these closely related plasmids.

Molecular and genetic characterization of Mu transposable elements in Zea mays: behavior in callus culture and regenerated plants.

Active Mutator lines of maize (Zea mays L.) have a high mutation rate and contain multiple hypomethylated 1.4-kb and 1.7-kb Mu transposable elements. Correlated with the inactivation of the Mutator system, these Mu elements cease to transpose and become more methylated. To determine whether the shock of tissue culture can affect Mutator activities, F1 progenies of outcrosses between active or inactive Mutator stocks and inbred line A188 were used to initiate embryogenic callus cultures. HinfI restriction digestion of genomic DNA isolated from 3-5-month-old cultures demonstrated that there is a very good correlation between the modification state of Mu elements in the cultures and the Mutator parent. Despite the dedifferentiation and rapid proliferation characteristic of tissue culture, the Mutator activity state is relatively stable during an extended tissue culture period. Cultures established from inactive Mutator lines were not reactivated; cultures established from active lines maintained a high Mu copy number, and most Mu elements remained unmodified. In contrast, weakly active Mutator parents gave rise to cultures in which Mu element modification could switch between low and high methylation during the culture period. Evidence for transposition was investigated with EcoRI digestion of genomic DNA isolated at different times during culture. The appearance of novel Mu-hybridizing fragments and a strong background hybridization are interpreted as evidence that transposition events occur during culture. Plants regenerated from such active cultures transmitted Mutator activity to their progeny.

Murine complement receptor gene family. II. Identification and characterization of the murine homolog (Cr2) to human CR2 and its molecular linkage to Crry.

CR2, a 145,000 to 150,000 Mr protein which binds specific breakdown products of C3, has been identified on the surface of both human and murine B cells. In order to understand the evolutionary relatedness of the human and murine proteins, we have used the coding sequences from the human CR2 gene to investigate those homologous sequences of murine Cr2. Human CR2 cDNA sequences were used as probes on a cDNA library derived from BALB/c spleen mRNA to identify cross-reacting cDNA sequences. A number of putative cDNA clones encoding murine Cr2 have been isolated and examined. DNA sequence analysis of these Cr2 cDNA clones indicates that they represent the murine homolog to human CR2. mRNA analysis with these Cr2 cDNA clones has revealed a transcription pattern similar to, but distinct from that seen for CR2. Whereas human CR2 coding sequences identify a single mRNA species of approximately 5 kb from human tonsillar mRNA, the murine counterpart identifies four transcripts from murine spleen of approximately 3, 5, 9 and 11 kb in size. The Cr2 cDNA clones which detect the four forms of spleen mRNA overlap in coding sequences and contain exons mapping to three colinear fragments as defined by EcoRI digestion. This suggests that the 3- 5-, 9-, and 11-kb mRNA forms arise by alternative splicing from a single gene. Use of these murine Cr2-specific cDNA clones to isolate their respective genomic sequences has allowed for the linkage of the 3' end of the Cr2 gene to the 5' end of the Crry gene, the evolutionary homolog to human CR1.

Normal Structure of the Gonadotropin-Releasing Hormone (GnRH) Gene in Patients with GnRH Deficiency and Idiopathic Hypogonadotropic Hypogonadism*

Idiopathic hypogonadotropic hypogonadism (IHH) is a genetic disorder in which a lack of hypothalamic GnRH secretion forms the basis for impaired gonadotropin release. Several lines of evidence, including an animal model of GnRH deficiency, suggest that an abnormality at the level of the GnRH gene might be responsible for the secretory defect in patients with IHH. We examined 13 patients (11 males and 2 females) with IHH documented by standard clinical and biochemical criteria, including apulsatile basal LH secretion which was reversed by administration of pulsatile GnRH. The structure of the GnRH gene in these patients was determined using Southern blot analyses. In every case EcoRI digestion revealed a 10-kilobase fragment including the entire GnRH gene, eliminating deletion of the gene as a cause of IHH. Multiple restriction enzymes (Xba I, BglII, BamHI, PstI, TaqI, RsaI, EcoRV, and BglI) and hybridization probes to exons 2 and 4 of the gene were used to search for polymorphisms or relatively small deletions or rearrangements in the GnRH gene. Polymorphisms were not identified, and deletions or rearrangements involving more than 200 basepairs were excluded. We conclude that a major rearrangement of the GnRH gene is not a common basis for IHH in humans.

Transposon Mutagenesis and Complementation of the Fructokinase Gene in Rhizobium leguminosamm biovar trifolii

Transposon Tn5 was used to generate a fructokinase mutation in Rhizobium leguminosarum biovar trifolii BAL. The section of the genome containing Tn5 was cloned into the EcoRI site of the vector pHC79 and isolated by direct selection on medium containing kanamycin and tetracycline. Total EcoRI digestion was used to obtain a single fragment containing Tn5 and flanking DNA sequences. The flanking DNA was used as a probe to isolate an intact fructokinase gene from a pLAFR1 cosmid clone bank of the parental strain. A cosmid showing homology to the probe was tri-parentally conjugated into the fructokinase-negative strain, complementing the mutation. The complemented mutant exhibited the wild-type phenotype, with an increase in fructokinase production presumably due to multiple copies of the gene.

Isolation and characterization of the Streptococcus mutans gtfD gene, coding for primer-dependent soluble glucan synthesis

Two glucosyltransferase genes from Streptococcus mutans GS-5, gtfB and gtfC, have been previously isolated and sequenced in this laboratory. In the present communication a third gtf gene, gtfD, was isolated and characterized. Isolation of the gene involved a novel procedure utilizing the integration plasmid pVA891. A peptide expressed by the 1.7-kilobase DNA fragment from strain NHS1 (containing deletions in both the gtfB and gtfC genes) was initially identified in a pUC18 clone bank with antiglucosyltransferase antibodies. This fragment was integrated into the GS-5 chromosome following ligation into pVA891 and transformation, yielding strain DP2. The vector together with one complete and one incomplete copy of the gtfD gene was removed from the chromosome of strain DP2 following EcoRI digestion, religation, and transformation of E. coli HB101. The resultant plasmid, pNH4, expressed glucosyltransferase S (GTF-S) activity. The enzyme was purified to near homogeneity and was shown to synthesize water-soluble glucan exclusively in a primer-dependent manner. The molecular mass (155 kilodaltons) and the kinetic parameters of the purified enzyme were similar to those observed for the GTF-S enzyme previously purified from culture fluids of strain GS-5. Insertional inactivation of the gtfD gene indicated that this gene is not required for in vitro sucrose-dependent adherence to smooth surfaces. Furthermore, inactivation of the gtfD gene in a gtfC gtfB mutant indicated that three distinct gtf genes involved in glucan formation are present on the S. mutans GS-5 chromosome. Southern blot analysis further suggested that the gtfD gene does not share demonstrable homology with the gtf genes from Streptococcus sanguis or Streptococcus sobrinus.

Distribution of a Secale cereale DNA repeat sequence among 25 Hordeum species

DNA of 61 accessions representing 25 Hordeum species was tested for homology to a highly repeated 120-bp sequence from Secale cereale (rye). Homology to the probe (pSC119) was detected in dot blots of all species except H. vulgare (cultivated barley) and its related species, H. agriocrithon and H. spontaneum. Hybridization patterns of Southern blots of restriction fragments demonstrated both intraspecific and interspecific variation in the organization of complex units of DNA having homology to the probe. For eight species, digestion of the DNA with BamHI gave ladder patterns characteristic of tandem arrays of 120-bp repeat units. For EcoRI, HindIII, and SacI digests, the hybridization patterns appeared to be highly conserved in the section Hordeum, except those for H. bulbosum, which were unique. A further set of patterns for these three enzymes was common among the remaining species of the genus. Thus, DNA hybridization with pSC119 generally gave patterns consistent with the current taxonomy of Hordeum species, except that H. bulbosum and H. vulgare were not shown to be closely related.Key words: Hordeum, repeated DNA sequences, pSC119, species variation.

Restriction analysis of λEMBL3 background recombinants: Occurrence of λ phages carrying a head to tail oriented left arm DNA sequence

Eight representative recombinant background clones of lambda EMBL3 were analysed using KpnI, BamHI, SalI, EcoRI and HindIII digestion. We found that lambda EMBL3 carries its own left arm in the BamHI cloning site. In the way, recombinant molecules were found to be generated which can grow on Escherichia coli strain NM539. In all cases analysed, the left arm DNA was inserted in a head to tail orientation. Seven clones carried a restored BamHI site at the cos site-BamHI site connection. In the region where the inserted left arm and the right arm were ligated, BamHI cloning produces a large palindromic sequence consisting of two polylinkers. This BamHI site was incompletely cleaved in all cases analysed. We assume that a part of the lambda DNA molecule in this region shows a cruciform structure prohibiting recognition or cleavage of this site by restriction endonuclease BamHI.

Cytosine methylation in the EcoRI site of active and inactive herpesvirus thymidine kinase promoters

The herpesvirus thymidine kinase ( tk) gene integrated in the human cell line, 2.1-a, can be inactivated by limited de novo methylation. All these TK − clones show partial EcoRI digestion of the recognition site (cGAATTCg) in the tk promoter in contrast to complete digestion of this site in the original cell line. Studies on well-defined substrates prepared in vitro showed that methylation of one cytosine in the EcoRI recognition sequence resulted in partial and methylation of both cytosines in severe inhibition of digestion by EcoRI. This characteristic was used to determine whether no, one or both cytosines in the EcoRI site of the tk promoter were methylated in various TK − clones derived from 2.1-a and in TK + clones re-expressing the gene after 5-azacytidine treatment. A high correlation was found between inactivity of the tk gene and methylation of only one of the two cytosines in the EcoRI recognition site. The results also show that the tk promoter can be active despite the presence of a methylated cytosine.

Allotypes of the constant region of the rabbit T cell receptor beta-2 chain.

Our laboratory previously reported that there was restriction fragment length polymorphism of TCR C beta genes in rabbits. EcoRI digests of DNA from different rabbits gave fragments of 9 and 6 kb (C beta a) or 14 and 6 kb (C beta b) that hybridized to a C beta cDNA probe. We also reported that the 9- and 14-kb types segregated as Mendelian traits and that there were allotypic differences in the first exon of the C beta 1 genes of C beta a and C beta b animals. Here we report the DNA sequence of the C beta 2 gene present in the 6-kb EcoRI fragment from a C beta b animal and compare the exon sequences with that of a cDNA from a C beta a animal. We find replacement changes in the first and third exons that probably represent allotypic forms of the rabbit C beta 2 gene. The genomic DNA 5' of exon 1 of both beta 1 and beta 2 contain alternating purine/pyrimidine repeat sequences. The genomic C beta 2 has an open reading frame of 69 amino acids in frame with exon 1 similar to a longer one previously found 5' of exon 1 of C beta 1. Further 5' of this region, rabbit C beta 1 and C beta 2 DNA sequences are only about 66% similar. Both the C beta 1 and C beta 2 sequences have two chi sequences; one in exon 1 with a perfect match and one in the intron downstream of exon 1 with one mismatch. Alternating purine/pyrimidine repeats and chi sequences found in rabbit C beta 1 and C beta 2 genes may have contributed to process(es) of gene duplication and/or conversion.

A repeated segment on the mouse Y chromosome is composed of retroviral-related, Y-enriched and Y-specific sequences.

We report the isolation and characterization of two recombinant clones containing DNA derived from the Y chromosome of the C57BL/10 inbred mouse strain. Both clones were isolated from a lambda phage library derived from a partial EcoRI digest of C57BL/10 male DNA using the murine retrovirus M720. Characterization of these clones showed they were derived from a repeated segment present on the C57BL/10J Y chromosome that contains sequences found elsewhere in the genome. In addition, one clone contained a sequence, designated YB10, that is unique to the Y chromosome and present in approximately 500 copies on the C57BL/10J Y chromosome. Analysis of Southern blots containing DNAs prepared from females and males of representative species from four subgenera of Mus probed with pYB10 and the 3'LTR from one of the Y-associated retroviruses (MuRVY) revealed that, with the exception of a single fragment observed in both female and male DNA of Mus saxicola, hybridization to pYB10 was observed only to male DNA of the species Mus spretus, Mus hortulanus, Mus musculus, Mus domesticus and Mus abbotti. In addition, the pattern and intensity of hybridization to YB10 and the MuRVY-LTR indicated that sequence of divergence was followed by amplification of Y chromosome sequences containing YB10 and MuRVY. The divergence and amplification occurred separately in each of the ancestral lineages leading to M. spretus, M. hortulanus, M. abbotti, M. musculus and M. domesticus. We suggest that acquisition and amplification of DNA sequences by the mammalian Y chromosome has contributed to its evolution and may imply that the mammalian Y chromosome is evolving at a faster rate than the rest of the genome.

Chorionic gonadotropin synthesis by human tumor cell lines: examination of subunit accumulation, steady-state levels of mRNA, and gene structure

A number of tumor cell lines have been examined that differentially produce human chorionic gonadotropin and the isolated α- or β-subunits. It has been demonstrated that all of the cell lines studied to date contain genes for both α- and β-subunits, indicating that differential and exclusive expression of one subunit is not the result of a particular cell line having lost the gene for the alternate subunit as a consequence of chromosome changes accompanying cell transformation. Because many of these established cell lines are aneuploid, it is also significant that no evidence was found for gene amplification in cell lines producing α-subunit at very high levels compared to those with very low level expression. Analysis of restriction endonuclease digests of tumor cell DNAs has demonstrated identical patterns for β-subunit in KpnI digests and KpnI/ HinIII double digests. Polymorphisms were observed for α-subunit in EcoRI and HinIII digests, but these did not correspond with expression of the α-subunit. Significant levels of either mRNA (as determined by dot blot and Northern transfer hybridization analysis) were accompanied by corresponding elevated levels of α- and β-subunits (as determined by radioimmunoassay), suggesting that regulation of subunit production most likely occurs at a pretranslational stage. However, there were apparent differences in the relative ratio of α- and β-subunits and their cognate mRNAs among the cell lines.

Alterations in c-abl gene methylation in cells transformed by phagocyte-generated oxidants

DNA from 10T1/2 cells transformed by activated neutrophils was analyzed for restriction length polymorphisms (RFLPs) in cellular homologues of retroviral oncogenes, and consistent RFLPs were found in MspI sites of the c-abl gene of all PMN-transformed cell lines. MspI digests probed with c-myc, v-Ki-ras, v-Ha-ras or v-mos showed no RFLPs, and none were observed in EcoRI, PstI, HindIII, BamHI, SmaI, Sau3a, MboI, HhaI, or TaqI digests probed with v-abl. Analysis of HpaII digests supports the conclusion that c-abl RFLPs result from differential methylation of the CCGG HpaII/MspI recognition sequence. MspI RFLPs in the c-abl gene may provide markers for oxidant-related genetic injury.

Molecular cloning and restriction endonuclease mapping of two strains of canine adenovirus type 2.

The DNA of a field isolate and of a vaccine strain of canine adenovirus type 2 (CAV-2) were analysed by digestion with several restriction endonucleases. The PstI restriction fragments of the field isolate (CAV-2 Glasgow) and the vaccine strain were cloned into the plasmid pBR322. Physical maps of the two viral genomes were constructed by molecular hybridization of PstI, EcoRI, SmaI, BamHI and KpnI digests of the viral DNA with the cloned PstI fragments. The restriction profile of CAV-2 Glasgow was shown to be virtually identical to those of the two prototype CAV-2 strains, Toronto A26/61 and Manhattan. However, the restriction fragment pattern of the vaccine strain of CAV-2 showed characteristic alterations, in particular additional sequences at or near the genome termini.

KRAS2 as a genetic marker for lung tumor susceptibility in inbred mice : Ryan J, Barker PE, Nesbitt MN, Ruddle FH. Department of Biology, Yale University, New Haven, CT 06510. J Natl Cancer Inst 1987;79:1351-7

An Eco-RI restriction fragment length polymorphism occurring in a DNA fragment containing the first exon of the murine KRAS2 gene was shown to correlate with the inherited susceptibility of inbred strains of mice to urethan (CAS: 51-79-6)-induced pulmonary adenomas. Eco-RI digestion of murine DNA yielded four KRAS2-specific fragments. Polymorphic variation occurred in the smallest molecular-weight fragment with alleles of either 0.70 or 0.55 kb in size. Genotyping of 14 inbred strains of mice revealed a correlation between KRAS2 Eco-RI polymorphic variation and the differential susceptibility among inbred strains to development of pulmonary adenomas. Strains with a high incidence of pulmonary adenomas, either spontaneously occurring or in response to carcinogen induction, had the 0.55-kb KRAS2 allele whereas adenoma-resistant strains had the 0.70-kb allele. Analysis of a series of recombinant inbred strains (AXB, BXA) that developed from reciprocal crosses between a highly susceptible strain (A/J) and a highly resistant strain (C57BL/6J) revealed a statistically significant threefold difference in lung tumor susceptibility on the basis of KRAS2 genotype. Further analysis of individual F2 mice of a C57BL/6 female X A/J male cross also demonstrated a threefold difference in tumor susceptibility on the basis of KRAS2 allelic variation.

Estrogen-Controlled Gene Expression: Induction of Two Estrogen-Responsive Genes in the Liver of Rainbow Trout (Salmo ǵairdneri)

Administration of 17β-estradiol to reproductively active male rainbow trout results in the induction of synthesis of two estrogen-responsive mRNA, vitellogenin (VG) and ULER2, to high levels. The complementary DNA (cDNA) of these two mRNA were prepared by molecular cloning. Two genomic DNA fragments generated by EcoRI digestion of trout genomic DNA showed hybridization to VG cDNA. A quantitative RNA dot blot hybridization method was adopted for the determination of levels of estrogen-responsive mRNA in livers of male trout after 17β-estradiol stimulation. This assay was used to determine the dose-response relationship and the time course of estrogen-responsive mRNA accumulation in juvenile fish.

In vitro studies on long-term stability of R plasmids in Escherichia coli K12.

The maintenance and stability in an E. coli K12 host of environmental isolates of R plasmids encoding gentamicin resistance during multiple passages in antibiotic-free and antibiotic-containing broth were investigated in regard to the conferred resistance phenotypes and the respective EcoRI digestion patterns. Only two plasmids belonging to the IncM group maintained stable endonuclease digestion patterns over a 15-month period in both of the media applied. Other members of this group revealed a considerable variability in their EcoRI digestion patterns, but a stability in their resistance determinants. Two IncM plasmids and an IncK plasmid exhibited partial resistance loss under nonselective conditions. The complete segregation of resistance determinants from IncOF plasmids resulted in a stably maintained cryptic core plasmid.