Evaluation of an automated DNA sequencing system developed in RIKEN

For the advancement of Human Genome Project, we have developed an automated DNA sequencing system HUGA-I. It is composed of several automated instruments and transfer robots connecting them. In this paper we describe the results of the performance evaluation test of HUGA-I. Although some of the system units showed good performances, the total performance of the HUGA-I was about 1/6 of the designed value. By revealing principal reasons of this poor performance, we would like to contribute to the automation in genome analysis, particularly in human genome analysis.Since the sequence technology advanced remarkably in these years, the system units of HUGA-I become older than those which are now commercially available and the throughput of it is out of our expectations. Nevertheless, we believe that it is meaningful to introduce the exact performance of HUGA-I and present the bottle neck points in the automating sequencing processes. Because, automation in the gene analysis is ultimately important, in particular for the analysis of large genomes such as the human genome. The aims of this paper are to introduce the results in performance evaluation of HUGA-I and to elucidate the bottle neck points in the automation of sequencing processes.The authors express their sincere thanks to Mr. Morisada Hayakawa and Mrs. Nobuko Kato for their technical asistance.     

The effects of tick numbers and intervals between infestations on the resistance acquired by guinea-pigs to adults of Rhipicephalus evertsi evertsi (Acari: Ixodidae)

Guinea-pigs were infested three times at short (1 week) intervals or long (10 week) intervals between successive infestations with low (1), medium (8) and high (15) numbers of adult Rhipicephalus evertsi evertsi. To compare the resistance developed by the hosts at short and long intervals between infestations, a challenge (fourth) infestation was performed with the high number of ticks. Resistance was assessed by comparing the mean weight of engorged female ticks that dropped from the hosts. Concentrations of beta globulins in the host sera were also monitored. Different tick Ioads (low, medium and high) and infestation intervals had a similar influence on the reduction in weight of the females which was between 60–70% after the final challenge. However, the concentration of beta globulins, was highest in the guinea-pigs exposed to medium and high numbers of ticks with long infestation intervals, probably because of an increase in the production of protective antibodies in response to more intensive antigenic challenge. The relationship between engorgement weight and beta globulin levels in host sera is discussed.     

Direct high-level secretion into the culture medium of tuna growth hormone in biologically active form byBacillus brevis

The characteristic features of theBacillus brevis system developed by us are very high productivity of heterologous proteins and very low extracellular proteinase activity. However, the production level of eucaryotic proteins with this system was generally one or two orders of magnitude lower than that of bacterial proteins. Therefore, we have explored methods for increasing the production efficiency as to animal proteins. Signal peptide modification was found to be very effective for high-level secretion of tuna growth hormone (tGH). Modification of the signal peptide with higher basicity in the amino terminal region and higher hydrophobicity in the middle region brought about a ten-fold increase in tGH production. Further elevation of the tGH yield to 240 mg/l was achieved by using a low proteinase mutant and a stable plasmid, and by culturingB. brevis under optimal conditions with the addition of some chemicals. Thus, biologically active tGH can be efficiently produced directly in the medium with thisB. brevis system.     

A novel oncogene, ost, encodes a guanine nucleotide exchange factor that potentially links Rho and Rac signaling pathways.

Transfection of NIH3T3 cells with an osteosarcoma expression cDNA library led to the appearance of foci of morphologically transformed cells which were found to harbor a novel oncogene, ost. The ost product was activated by truncation of the N-terminal domain of the ost proto-oncogene and was highly tumorigenic in nude mouse assays. The proto-ost cDNA, isolated subsequently, encodes a predicted protein of 100 kDa containing DH (Db1 homology) and PH (pleckstrin homology) domains. Ost is mainly phosphorylated on serine and localized in the cytoplasm. Purified Ost protein catalyzed guanine nucleotide exchange on RhoA and Cdc42 among the Rho and Ras family members tested, indicating that Ost can activate these small GTP-binding proteins. Ost did not detectably associate with RhoA or Cdc42, but interacted specifically with the GTP-bound form of Rac1, suggesting that Ost can function as an effector of Rac1. These results suggest that Ost is a critical regulatory component which links pathways that signal through Rac1, RhoA and Cdc42. Of the tissues examined, expression of ost was the highest in brain and could be localized to neurons and alpha-tanycytes, suggesting that Ost may participate in axonal transport in these specialized cells.     

Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms.

A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately adjacent from the polymorphic site of interest. Using the Klenow fragment of E. coli DNA polymerase I or the modified T7 DNA polymerase (Sequenase), the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates. Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format. This paper describes biochemical features of this method in detail. A semi-automated version of the method, which we call Genetic Bit Analysis (GBA), is being used on a large scale for the parentage verification of thoroughbred horses using a predetermined set of 26 diallelic polymorphisms in the equine genome.