Robert L. Ratliff

Isolation and molecular characterization of a highly polymorphic centromeric tandem repeat in the family falconidae

An abundant tandem repeat has been cloned from genomic DNA of the merlin (Falco columbarius). The cloned sequence is 174 bp in length, and maps by in situ hybridization to the centromeric regions of several of the large chromosomes within the merlin karyotype. Complementary sequences have been identified within a variety of falcon species; these sequences are either absent or in very low copy number in the family Accipitridae. The cloned merlin repeat reveals highly polymorphic restriction patterns in the peregrine falcon (Falco peregrinus). These polymorphisms, which have been shown to be stably inherited within a family of captive peregrines, can be used to differentiate the Greenland and Argentina populations of this endangered raptor species.

High-speed DNA sequencing: An approach based upon fluorescence detection of single molecules

We are developing a laser based technique for the rapid sequencing of large fragments (approximately 40 kb) of DNA based upon the detection of single, fluorescently tagged nucleotides cleaved from a single DNA fragment. We have demonstrated significant progress on several of the important steps of this technique. The projected rate of sequencing is several hundred bases per second which is orders of magnitude faster than existing methods. Once developed, this technology could be utilized by investigators for rapid sequencing of genetic material from virtually any source.

Circular dichroism spectroscopy of DNA.

Publisher Summary Circular dichroism (CD) measurements are used to study the conformations of nucleic acids in solution. The reliance on CD spectroscopy to study DNA conformations has stemmed from the sensitivity and ease of CD measurements, the nondestructive nature of such measurements, the fact that conformations can be studied in solution, and the requirement for relatively small amounts of material. Although detailed structural information, such as from X-ray crystallography or NMR spectroscopy, is not available from CD spectra, the CD spectrum of DNA in solution can provide a reliable determination of its overall conformational state when compared with the CD spectra of reference samples. Moreover, CD spectroscopy is applicable to a wide range of samples, including those that are difficult to crystallize or to obtain at high concentrations. The chief practical application of CD spectroscopy to the study of DNA structures has been by making empirical comparisons with the CD spectra of known structures. The chapter provides examples from laboratory of CD spectral changes in the near ultraviolet range that illustrate the CD characteristics of structural transitions in natural and synthetic DNAs.

Terminal deoxynucleotidyl transferase in the diagnosis of leukemia and malignant lymphoma.

Neoplastic cells from 253 patients with leukemia and 46 patients with malignant lymphoma were studied for the presence of terminal deoxynucleotidyl transferase (TdT) by biochemical and fluorescent antibody technics. TdT was detected in circulating blast cells from 73 of 77 patients with acute lymphoblastic leukemia, 24 of 72 patients with chronic myelogenous leukemia examined during the blastic phase of the disorder and in cell suspensions of lymph nodes from nine of nine patients with diffuse lymphoblastic lymphoma. Blast cells from six of 10 patients with acute undifferentiated leukemia were TdT positive, but the enzyme was found in only two of 55 patients with acute myeloblastic leukemia. TdT was not detected in other lymphocytic or granulocytic leukemias or in other types of malignant lymphomas. The fluorescent antibody assay for TdT permits rapid and specific identification of the enzyme in single cells. The TdT assay is clinically useful in confirming the diagnosis of acute lymphoblastic leukemia, evaluating patients with blastic chronic myelogenous leukemia, and distinguishing patients with lymphoblastic lymphoma, whose natural history includes rapid extranodal dissemination, from patients with other poorly differentiated malignant lymphomas.

Cell-cycle-dependent variations of deoxyribonucleoside triphosphate pools in chinese hamster cells

Abstract Variation of levels of the four deoxyribonucleoside triphosphate pools has been examined as synchronized Chinese hamster cells complete mitosis and traverse the cell cycle in preparation for division. The results indicate that; 1. 1. Mitotic cells have the largest pools of dATP, dGTP, and dTTP and that all four deoxyribonucleoside triphosphates are degraded as cells exit from mitosis. 2. 2. The pool of dCTP is maximal in late S-early G2, and degradation begins in G2. 3. 3. G1 cells have little or no deoxyribonucleoside triphosphates. 4. 4. The pool size of all four deoxyribonucleoside triphosphates increases just prior to initiation of DNA synthesis and increases throughout S. 5. 5. Cells treated with hydroxyurea in G1 accumulate dTTP, dCTP, and dGTP at the time that cells would normally have initiated DNA synthesis, but accumulation of dATP is completely inhibited as is initiation of DNA synthesis. 6. 6. The four deoxyribonucleoside triphosphates are not present in equimolar concentrations, pools in mid-S being 10 pmoles 10 6 cells , 27 pmoles 10 6 cells , 104 pmoles 10 6 cells , and 76 pmoles 10 6 cells for dGTP, dATP, dTTP, and dCTP, respectively. The pools of dGTP, dATP, dTTP, and dCTP are sufficient to support DNA synthesis for 1.0, 1.3, 5.2, and 3.8 min, respectively.

Rapid DNA sequencing based upon single molecule detection

We are developing a laser-based technique for the rapid sequencing of 40-kb or larger fragments of DNA at a rate of 100 to 1000 bases per second. The approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA fragment into a flowing sample stream, and detection of individual fluorescently labeled bases as they are cleaved from the DNA fragment by an exonuclease. The ability to sequence large fragments of DNA will significantly reduce the amount of subcloning and the number of overlapping sequences required to assemble megabase segments of sequence information.

Extension of the four-stranded intercalated cytosine motif by adenine.adenine base pairing in the crystal structure of d(CCCAAT).

The crystal structure of d(CCCAAT), refined at 2.0 Å resolution, shows a four stranded molecule in which two parallel duplexes intercalate with opposite polarity, using cytosine•protonated cytosine base pairs. The intercalation motif in this structure is extended by adenine•adenine base pairs. Two topologically distinct broad grooves are found in the lath-like central part of the molecule with the phosphate groups on one side bent over towards each other, stabilized by bridging water molecules. At the 3′ends, two arrangements of intermolecular A•A•T base triplets are found, involving both asymmetric and symmetric A•A base pairs joined to thymine residues by Watson-Crick and reverse Hoogsteen base pairing, respectively.

Biosynthesis of poly(γ-glutamylcysteinyl)glycines in cadmium-tolerant Datura innoxia (Mill.) cells

The presence of γ-carboxamide linkages in poly(γ-glutamylcysteinyl)glycines ((γEC)nG) suggests that these polypeptides are likely to be the products of a biosynthetic pathway. However, the in vivo deamidation of proteins can result in the introduction of γ-carboxamide linkages. Synthetic oligodeoxynucleotide sequences were therefore used as probes to hydridize to any mRNA sequences which could encode such proteins. These probes did not hybridize to any mRNA sequences from cells grown either in the presence or absence of Cd, confirming that these metal-binding polypeptides are not directly encoded by structural genes. The kinetics of induction of synthesis of (γEC)n G following exposure to Cd, was studied using reverse phase HPLC of extracts from cells grown in the presence of [35S]cysteine. These data indicate that the shorter polypeptides are substrates for the synthesis of longer forms. Induced synthesis of (γEC)2G and (γEC)3G was detected 5 min after exposure of cells to Cd. This rapid response implies that the pathway is regulated, at least initially, at a post-translational level. Observed insensitivity of this response to cycloheximide further supports this conclusion.

Deproteinization with phenol of alternating polydeoxyadenylate-deoxythymidylate and other DNA-like polymers

Abstract It has been reported that the deoxythymidylate-deoxyadenylate-rich satellite DNA from the land crab is lost into the phenol phase during phenol extraction of tissues from that organism. Others report that alternating poly deoxyadenylate-deoxythymidylate is lost into the phenol phase during deproteinization. The reasons for this selective loss were investigated, and it was shown that the high NaCl concentrations used during extraction are responsible. A phenol extraction method that gives quantitative recovery of alternating poly deoxyadenylate-deoxythymidylate and other enzymically synthesized polymers is described.

Conformations of poly(d(A-T-T))-poly(d(A-A-T)).

The sodium salt of poly[d(A-T-T)]·poly[d(A-A-T)] is observed in oriented and partially crystalline fibers to have the classical B -DNA molecular conformation at relative humidities >85% and the B -type D -form hitherto observed only for poly[d(Pur-Pyr)]·poly[d(Pyr-Pur)] DNAs, at relative humidities of zero to 85%. No A -type conformation is observed.