Dye structure affects Taq DNA polymerase terminator selectivity.

All DNA sequencing methods have benefited from the development of new F667Y versions of Taq DNA polymerase. However, terminator chemistry methods show less uniform peak height patterns when compared to primer chemistry profiles suggesting that the dyes and/or their linker arms affect enzyme selectivity. We have measured elementary nucleotide rate and binding constants for representative rhodamine- and fluorescein-labeled terminators to determine how they interact with F667 versions of Taq Pol I. We have also developed a rapid gel-based selectivity assay that can be used to screen and to quantify dye-enzyme interactions with F667Y versions of the enzyme. Our results show that 6-TAMRA-ddTTP behaves like unlabeled ddTTP, while 6-FAM-ddTTP shows a 40-fold reduction in the rate constant for polymerization without affecting ground-state nucleotide binding. Detailed mechanism studies indicate that both isomers of different fluorescein dyes interfere with a conformational change step which the polymerase undergoes following nucleotide binding but only when these dyes are attached to pyrimidines. When these same dyes are attached to purines by the same propargylamino linker arm, they show no effect on enzyme selectivity. These studies suggest that it may be possible to develop fluorescein terminators for thermocycle DNA sequencing methods for polymerases that do not discriminate between deoxy- and dideoxynucleotides.

Slow rate of phosphodiester bond formation accounts for the strong bias that Taq DNA polymerase shows against 2',3'-dideoxynucleotide terminators.

Taq and T7 DNA polymerases have become basic molecular biology "tools" for DNA sequence analysis. However, Taq, unlike T7 DNA polymerase, is strongly biased against the incorporation of 2',3'-dideoxynucleotide triphosphates (ddNTPs) indicating very different substrate selectivities. Equilibrium binding and rate constants were measured for 2',3'-ddNTPs as well as for several other 3'-substituted terminators and compared to 2'-deoxynucleotide substrates (dNTPs). In steady-state experiments, Taq Pol I was strongly biased in favor of dATP1 over ddATP incorporation by about 700 to 1, in contrast to T7 DNA polymerase which showed a preference of only about 4 to 1. Manganese reduced but did not eliminate selectivity against 2',3'-ddNTPs. Transient kinetic traces indicated different rate-limiting steps for substrate and terminator incorporation. Further mechanistic studies showed that the binding constants for substrates and terminators were equivalent. However, the rate constants for phosphodiester bond formation for 2',3'-ddNTPs were 200-3000-fold lower than for dNTPs. Alternative terminators showed only slight improvements. The data were consistent with a model in which both substrates and terminators undergo ground-state binding followed by formation of a tight-binding Enz.DNA.Nucleotide complex. Immediately after complex formation, substrates undergo a rapid nucleoside phosphoryl transfer reaction. However, the reaction rates for terminators were slower presumably due to misalignment of reactive groups in the active site. Thus, the strong bias that Taq DNA polymerase shows against terminators is due to a very slow "chemistry" step. Such a strong bias has several kinetic consequences for DNA sequence patterns. These consequences are discussed in the text.

Construction of a chimeric antibody with therapeutic potential for cancers which overexpress c-erbB-2.

We describe the chimerization of a monoclonal antibody directed against the c-erbB-2 protein using a novel PCR method for cloning immunoglobulin variable region genes. We also describe the characterization of the chimera and show its potential use for treating cancers which overexpress the c-erbB-2 protein. The genomic DNA fragments of heavy and light chain variable genes were cloned by PCR using uniquely designed primers which allowed for isolation of genes containing functional promoters, signal and coding sequences. The chimeric genes were then constructed by linking variable regions of murine genes to human constant gamma 1 and kappa genes. Expression of the chimeric immunoglobulin genes resulted in production of properly assembled chimeric antibody with improved biological properties.

Serological evaluation of Escherichia coli-expressed human T-cell leukemia virus type I env, gag p24, and tax proteins.

Three proteins (env, gag, and tax) encoded by the human T-cell leukemia virus type I (HTLV-I) genome were cloned and expressed in Escherichia coli. The env protein contained a substantial part of the gp46 domain and a majority of the p21e domain. The gag protein contained all of p24 and portions of p19 and p15. In addition to these two structural proteins, a full-length tax (p40X) construct was obtained. All three recombinant proteins were purified to near homogeneity. When used in an immunoblot assay, the three recombinant proteins detected antibodies in more HTLV-I antibody-positive patient sera than did the corresponding native proteins. Antibodies to at least two of these three different gene products were detected in 98.4% of adult T-cell leukemia patients, 100% of HTLV-I-associated myelopathy patients, 97.4% of asymptomatic carriers, and 94% of uncharacterized HTLV-I-positive patients. Antibody to recombinant tax was found in 4.9% of adult T-cell leukemia patients, whereas antibody to recombinant env could not be detected. These recombinant proteins from three different gene products may be useful in detecting or confirming the presence of antibodies to HTLV-I.

Cytoplasmic microtubule proteins of the embryo of Drosophila melanogaster.

We have been able to purify, in bulk, the cytoplasmic microtubule proteins of eggs and embryos of Drosophila melanogaster by means of in vitro self-assembly of microtubules from subunits present in a high-speed supernatant fraction of eggs or embryos. This provides the first successful application of this method to purification of microtubule protein from a source other than vertebrate brain, and the first purification of insect microtubule proteins. Our electron micrographs show that the in vitro assembled microtubules are morphologically typical and apparently are comprised of the expected 13 protofilaments. The protein we obtain from such preparations binds [3H]colchicine and has a sedimentation value of 6.4 S-6.9 S which is close to the predicted value for microtubule protein dimer. Both alpha- and beta-microtubule proteins are evident in sodium dodecyl sulfate polyacrylamide electropherograms of the isolated proteins. The apparent molecular weights of these species on dodecyl sulfate polyacrylamide gels are 54,000 and 52,000, respectively. These values as well as the amino acid composition and N-terminal methionine of the Drosophilia proteins are very closely comparable to microtubule proteins from other, unrelated organisms.

Microtubule protein pools in early development.

Microtubule protein pools have been demonstrated to exist in unfertilized eggs and the early embryonic stages of several organisms. The microtubule pool of the sea urchin embryo is constant in size (about 0.4% of the total embryo protein) throughout early development. Protein withdrawn from this pool for organelle assembly is replaced by new synthesis. Eggs and embryos of Drosophila similarly contain a pool of microtubule proteins (larger than or equal to 0.4% of the total embryo protein, congruent to 3% of the soluble protein), which is constant in size throughout early development. The Drosophila egg microtubule proteins are easily purified by self-assembly in vitro of microtubules, and are similar to microtubule proteins from other organisms in molecular weight and other properties. Synthesis of microtubule proteins in sea urchin embryos is supported by oogenetic mRNA. This appears also to be the case in molluscan (Ilyanassa) embryos. It is not known whether Drosophila embryos synthesize microtubule proteins during the early stages of development.