The isolation and characterization of the chicken lysozyme and ovomucoid gene.
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Animals↗
Biomedical subjects
N Blin
Publications and source records attributed to N Blin.
Base distribution in the coding and noncoding regions in the rDNA of Lytechinus variegatus.
The EcoRI restriction endonuclease cleaves rDNA repeat units of the sea urchin Lytechinus variegatus into four fragments. The G + C contents of all four cloned restriction fragments were determined by ispycnic analysis. Electron microscopic denaturation mapping of one of the fragments allowed alignment of the denaturation pattern with the restriction map and correlation with previously reported transcriptional data. From these results the base distribution in the spacer region and regions coding for rRNAs was derived.
Base Composition↗
Isolation and characterization of the chicken ovomucoid gene.
The chicken ovomucoid gene has been isolated by screening a chicken DNA library with a plasmid containing ovomucoid mRNA sequences. Twelve recombinant phages carrying ovomucoid mRNA sequences were isolated. Two of them, extending farthest into the 5' and 3' direction respectively, were characterized by restriction mapping and Southern hybridization as well as by electron microscopic analysis of hybrids between the cloned DNA and ovomucoid mRNA. Seven intervening sequences interrupt the ovomucoid mRNA sequence in chromosomal DNA. From these data a minimal size of 5.6 kb can be estimated for the length of the ovomucoid gene.
Animals↗
Organization of the ribosomal RNA gene cluster in Lytechinus variegatus. Restriction analysis and cloning of restriction fragments.
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Animals↗
Arrangement of coding and intervening sequences of chicken lysozyme gene.
Hybrid phages that contain chicken lysozyme gene sequences have been isolated from a chicken DNA library. Two overlapping clones covering a region of 22 kilobase pairs around this gene have been studied by restriction mapping. Southern hybridization, and electron microscopic analysis of hybrids between lysozyme mRNA and the cloned cellular DNA. Three intervening sequences interrupt the lysozyme structural gene. The cellular gene is at least 3.9 kilobases long, about 6 times the length of the structural gene.
Animals↗
The ribosomal RNA gene region in the sea urchin Lytechinus variegatus.
A fine structure map of the ribosomal gene region of the sea urchin Lytechinus variegatus has been generated. The ribosomal DNA density satellite has been analyzed with restriction enzymes and by hybridization to urchin 18S and 26S ribosomal RNA (rRNA). The L. variegatus rRNA repeats show heterogeneity on the nontranscribed spacer. Minor bands containing transcribed spacer sequences are observed. One interpretation of these bands would require both transcribed spacer heterogeneity and defective ribosomal genes retained among the rRNA gene repeats.
Animals↗
A denaturation map of sea urchin ribosomal DNA.
The ribosomal RNA genes from the sea urchin Lytechinus variegatus have been studied with the electron microsocope using the technique of denaturation mapping. A repeating pattern of denatured regions was found with an average repeat length of 3.87 +/- 0.24 mum. This corresponds to a DNA sequence of approximately 12,000 base pairs with a molecular weight of 8 X 10(6) daltons.
Animals↗
Isolation and some properties of a mammalian ribosomal DNA.
The DNA coding for 28 S and 18 S ribosomal RNA, including the spacer regions, has been isolated from calf (Bos taurus) thymus gland. The method used included shearing of the total DNA to a highly homogeneous size population, selective heat denaturation and S1 nuclease treatment to remove single stranded DNA. Repeated centrifugation on density gradients yields a 140-fold purified rDNA fraction with a GC content of 61.2% Eco RI nuclease cleaves this DNA into two fragments of 16.4 and 4.9 X 10(6) daltons. Hybridization of these fragments with 28 S and 18 S rRNA shows that the 28 S coding sequence is located mostly on the 4.9 X 10(6) dalton fragment, while both the 16.4 and 4.9 X 10(6) dalton fragments contain the 18 S sequence. The data indicate that the ribosomal RNA gene has a repeat unit of 21.3 X 10(6) daltons which includes a nontranscribed spacer of about 12.5 X 10(6) daltons.
Animals↗
A general method for isolation of high molecular weight DNA from eukaryotes.
A new method for isolation of high molecular weight DNA from eukaryotes is presented. This procedure allows preparation of DNA from a variety of tissues such as calf thymus or human placenta and from cells which were more difficult to lyse until now (e.g. Crypthecodinium cuhnii, a dinoflagellate). The DNA obtained in such a way has an average molecular weight of about 200 X 10(6) d and contains very few, if any, single strand breaks.
Animals↗
Isolation of large molecular weight DNA from agarose gels for further digestion by restriction enzymes.
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Centrifugation, Isopycnic↗
Cloning and organization of genes for 5S ribosomal RNA in the sea urchin. Lytechinus variegatus.
A 1.35-kb EcoRI fragment of Lytechinus variegatus DNA containing a single 5S rRNA gene has been cloned into the plasmid vector pACYC184. Four clones from different transformation experiments contain 5S rDNA inserts of about the same size and have the same restriction enzyme digestion patterns for the enzymes HaeIII, HinfI, HhaI, and AluI. One EcoRI site near the HindIII site of the plasmid vector pACYC184 is missing in all the four clones. By DNA sequencing, the missing EcoRI ws found to be EcoRI site, d(AAATTN)d(TTTAAN) in pLu103, one of the four 5S rDNA clones. The structure of pLu103 was determined by restriction mapping and blot hybridization. Three restriction fragments, 1.0-kb HaeIII/HaeIII, 0.375-kb AluI/AluI and 0.249-kb MboII/MboII, which contain the 5S rRNA coding region, have been subcloned into the EcoRI site of the plasmid pACYC184. The organization of 5S rRNA genes in the sea urchin genome was also investigated. It was found that restriction endonuclease HaeIII has a single recognition site within each 5S rDNA repeat, and yields two fragment lengths, 1.2 and 1.3 kb. The behavior of these 5S rRNA genes when total L. variegatus DNA is partially digested with HaeIII is consistent with an arrangement of 5S rRNA genes in at least two tandemly repeated, non-interspersed families. Both the coding region and spacer region of the 5S rRNA gene in pLu103 hybridize to 1.2 and 1.3-kb rDNA families. This indicates that the cloned EcoRI fragment of 5S rDNA in pLu103 represents one single repeat of 5S rDNA in the genome.
Animals↗
Simultaneous isolation of high molecular weight RNA and DNA from limited amounts of tissues and cells.
A simple method is described for the simultaneous isolation of both DNA and RNA from tissues and cultured cells obtainable in limited quantities only. The method is based on a suitable combination of steps designed for preparations of high molecular weight nucleic acids in cases when restricted amounts of tissues like small-sized and unique biopsies of tumors are available for studies of gene organization and expression. Using this protocol, undegraded total RNA suitable for Northern blot analysis and high molecular weight DNA for Southern blots was obtained from various sources (mammary and colon carcinomas, meningiomas, colonic and placental tissue, and several cell cultures).
Blotting, Southern↗
Expression of the c-erbB2 proto-oncogene in male breast carcinoma: lack of prognostic significance.
Increase of gene activity of the proto-oncogene erbB2 which codes for the transmembrane kinase receptor p185erbB2 has been observed in > 30% of female breast and gynecological carcinomas. This overexpression was shown to be correlated with poor prognosis. We have investigated 38 samples of carcinomas of the male breast for p185erbB2 expression by using tumor thin sections and a monoclonal antibody. The immunostaining was compared to clinical data to assess a possible prognostic value of this parameter. Although most cases were immunopositive (36/38), no correlation to tumor grading and survival spans was notable. Therefore, erbB2 activity fails to add a new prognostic parameter in male breast carcinomas.
Biomarkers, Tumor↗
Observations on leukocytes from patients with severe familial hypercholesterolemia.
Differences in light scatter and fluorescence emission of blood leukocytes from patients with familial hypercholesterolemia (FH) and normal patients incubated with oil red O and r(NBD-Cl)-acetyl-LDL were observed by flow-through cytofluorometry and used to distinguish a subclass of leukocytes. The appearance of this subpopulation of leukocytes in FH apparently correlated with the excessive hypercholesterolemia. In addition, microscopic evaluation of blood smears suggested the formation of small cytoplasmic vacuoles in a population of FH monocytic cells. While these are preliminary observations on a small number of patients, they warrant further study. However, the use of lipophilic dyes and fluorescent acetyl-LDL in combination with flow cytometry may be another useful means for evaluation of FH.
Azo Compounds↗
Assessing chromosome 22 microdeletions in meningioma with new polymorphic DNA markers.
In an approach to identify new candidates for tumor related genes, a set of established and new polymorphic DNA markers (simple sequence repeats) was used in menigioma samples to test for deletions within human chromosome 22. For this purpose, cytogenetically well characterized tumors were applied and those with monosomy #22 were omitted; only disomic material was analyzed. When comparing DNA from peripheral blood and tumor tissues using markers spaced throughout the long arm (22q) no allelic loss was observed indicating that, in disomic samples, deletions are present as microdeletions undetectable by the marker spacing chosen in this study, or that they do not occur frequently.
Biomarkers, Tumor↗
Assessing genetic heterogeneity of renal cell tumors.
Renal cell tumors display a highly variable morphology which is also reflected at the genomic level. Such heterogeneity was at first monitored by cytogenetic means (numerical and structural chromosomal aberrations); in the meantime, more refined molecular techniques allow the assessment of DNA losses or gains in metaphase chromosomes or tissue sections. Moreover, genomic instability can be monitored using microsatellite probes. All these methods document specific characteristics of certain renal cell tumor types, e.g. telomeric associations in chromophobe carcinomas or oncocytomas, typical losses in 3p in clear cell carcinomas or trisomy 7 in renal cell adenomas and carcinomas. Next to examples demonstrating these alterations the Heidelberg classification of renal cell tumors that is based on genomic observations is discussed.
Biopsy↗
Tumor-specific methylation patterns of erbB2 (HER2/neu) sequences in gastro-intestinal cancer.
To determine whether the sporadically occurring amplification of the oncogene erbB2/HER2 in gastrointestinal carcinomas is associated with additional changes of this sequence, DNA from 17 colorectal and 5 stomach carcinomas was analyzed for copy number, sequence rearrangement and DNA methylation by Southern blot hybridization. Amplification was detected in two cases. By applying the isochizomers Hpall and Mspl we tested for alterations in the DNA methylation status. Whereas in colon tumors with non-amplified erbB2 this status was unchanged, one case with erbB2 amplification showed additional MspI bands indicating a methylation of the amplified gene sequences. In stomach carcinoma, however, we detected differences between tumor and mucosa samples but not between amplified and non-amplified tumor samples. Independent of the DNA methylation status, significant amounts of the erbB2 oncoprotein were detected in the cases with gene amplification; weaker immunostaining of erbB2 was also seen in a few additional tumors.
Blotting, Southern↗
Differential activity of two oncogenes from chromosome #7 in human glioblastoma cell lines.
Human glioblastoma cell lines are known to develop polysomy of cytogenetically intact chromosomes #7 and overexpression of the erbB oncogene (7p12-p14) at a level even higher than is to be expected from the number of #7 chromosomes. The met oncogene, however, which is also located on chromosome #7 (7q31-q32), was shown not to be overexpressed in a panel of 7-polysomic glioblastoma cell lines overexpressing erbB. Molecular analysis of the cell line HeRo gave proof that there is no detectable amplification or rearrangement of the erbB gene which could be responsible for its overexpression. These findings favor the assumption of differential regulation of the met and erbB oncogenes, e.g. by means of insufficient activity of a trans-acting erbB suppressor gene possibly located on a chromosome with a low copy number.
Chromosome Aberrations↗