Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method

Homologous alignment cloning (HAC) is a rapid method of molecular cloning that facilitates low-cost, highly efficient cloning of polymerase chain reaction products into any plasmid vector in approximately 2 min. HAC facilitates insert integration due to a sequence alignment strategy, by way of short, vector-specific homology tails appended to insert during amplification. Simultaneous exposure of single-stranded fragment ends, utilising the 3′→5′ exonuclease activity of T4 DNA polymerase, creates overlapping homologous DNA on each molecule. The exonuclease activity of T4 polymerase is quenched simply by the addition of EDTA and a simple annealing step ensures high yield and high fidelity vector formation. The resultant recombinant plasmids are transformed into standardE. colicloning strains and screened via established methods as necessary. HAC exploits reagents commonly found in molecular research laboratories and achieves efficiencies that exceed conventional cloning methods, including another ligation-independent method we tested. HAC is also suitable for combining multiple fragments in a single reaction, thus extending its flexibility.

Independent synaptic behavior of sister chromatids in Coprinus cinereus

Sister and nonsister chromatids behave in distinct ways during meiosis. We have shown that homologous synapsis does not require the presence of sister chromatids. In mutants that fail to undergo premeiotic DNA replication, synaptonemal complex of normal appearance is observed in diploid nuclei, but not in triploid nuclei. Our results indicate that homologous alignment does not depend on DNA replication. In addition, the differences observed in diploid and triploid nuclei indicate that sister chromatids can exhibit independent synaptic behavior, although ordinarily, they are constrained to act as a single unit. Key words: Coprinus cinereus, meiosis, synaptonemal complex, meiotic mutants, DNA replication, triploid nuclei.

Synaptonemal complex spreading in Allium. II. Tetraploid A. vineale

In tetraploid Allium vineale four homologous axes are closely aligned in unsynapsed regions at early zygotene. This alignment is brought about by intercalary and terminal associations. The intercalary association sites are possibly targets of forces for long distance attraction of homologues and potential pairing initiation sites. The terminal associations are mediated by dense spherules and result possibly from the attachment of the telomeres to the nuclear envelope. In pachytene the alignment is abolished and the four axes are synapsed two by two, resulting in bivalents or, owing to partner switches in the synapsed axes, in quadrivalents. From the number of partner switches per configuration the number of pairing initiation sites is estimated. Homologous alignment and synapsis are discussed, comparing them with the conditions in a triploid species, which were described in a previous paper.Key words: Allium vineale, synaptonemal complex, polyploids, chromosome pairing, meiosis.

The Primary Structure Of Crotalase, A Thrombin-Like Venom Enzyme, Exhibits Strong Homology With Kallikrein

Edman degradation of crotalase and its tryptic and CNBr fragments yielded an N-terminal amino acid sequence, Val- Ile-Gly-Gly-Asp-Glu-Cys-AsN-Ile-AsN-Glu-His-Arg-Phe-Leu-Val- Ala-Leu-Tyr-Asp-Tyr-Trp-Xxx-GlN-Xxx-Phe-rLeu-, and internal sequences, -Leu-Ile-Arg-Leu-AsN-Lys-Pro-Val-Ser-Tyr-Ser-Glu- His-Ile-Ala-Pro-Leu-Ser-Leu-Pro-Ser-Ser-Pro-Pro-Ile-Val-Gly- Ser-Val-Cys-Arg-Ala-Met-Gly-Trp-Gly-GIN-Thr-Thr-Ser-Pro-GIN- Glu-Thr-Leu-Pro-Asp-Val-Pro-His-Cys-Ala-AsN-Ile-AsN-Leu-Leu- Asp-Tyr-Glu-Val-Cys- and -Ser-Val-GIN-Phe-Asp-Lys-Glu-GIN- GIN-Arg-. The longer internal sequence aligns homologously with positions 105-168 of chymotrypsinogen with gaps, each 5 residues long, following Pro 128 and GIN 143. No homologous alignment has been found for the shorter internal sequence. These findings led to experiments which demonstrated that crotalase has specific enzymatic properties resembling kallikrein.

Specific end-to-end attachment of chromosomes in Ornithogalum virens

C-banding of nonhomologous chromosomes in haploid generative nuclei of Ornithogalum virens (n = 3) reveals a high degree of specificity with respect to end-to-end connexions. The centromeric end of chromosome 2 preferentially associates with the centromeric end of chromosome 3 and the telomeric end of chromosome 3 associates preferentially with the telomeric end of chromosome 1. This same association of nonhomologous chromosomes persists in prophase nuclei of diploid root tips. In addition, the telomeric ends of the 2 chromosome 2s are connected to one another as are the centromeric ends of the chromosome 1s. This results in a ring of chromosomes in which homologues lie opposite one another. Centromeric ends lie on one side of the nucleus and telomeric ends on the other. It is proposed that this specific association of chromosome ends reflects an order which was probably established at the preceding anaphase or telophase and which persists throughout interphase. The suggestion is made that the proximity of homologous ends and consequently homologous alignment may facilitate initiation of pairing at meiosis.