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.     

A Mucor pusillus mutant defective in asparagine-linked glycosylation.

A Mucor pusillus mutant defective in asparagine-linked glycosylation was found in our stock cultures. This mutant, designated 1116, secreted aspartic proteinase (MPP) in a less-glycosylated form than that secreted by the wild-type strain. Analysis of enzyme susceptibility, lectin binding, and carbohydrate composition indicated that this mutant secreted three glycoforms of MPPs, one of which contained no carbohydrate; the other two had truncated asparagine-linked oligosaccharide chains such as Man0-1GlcNAc2. Further analysis using oligosaccharide processing inhibitors, such as castanospermine, 1-deoxynojirimycin and N-methyldeoxynojirimycin, suggested that MPPs in the mutant were glycosylated through a transfer of the truncated lipid-linked oligosaccharides, Man0-1GlcNAc2, to the MPP protein but not through an aberrant processing. In addition, genetic studies with forced primary heterokaryons indicated that the mutation in strain 1116 was recessive.     

Systemic amyloidosis in transgenic mice carrying the human mutant transthyretin (Met 30) gene

To analyze the pathologic processes of amyloid deposition in type I familial amyloidotic polyneuropathy (FAP), mice were made transgenic by introducing the human mutant transthyretin (TTR) gene(MT-hMet 30). An inbred strain of mouse, C57 BL/6, was chosen. Transgenic mice were killed using ether anesthesia at 3-mo intervals up to 24 mo after birth. In these transgenic mice, amyloid deposition started in the gastrointestinal tract, cardiovascular system, and kidneys and extended to various other organs and tissues with advancing age. The pattern of amyloid deposition was similar to that observed in human autopsy cases of FAP, except for its absence in the choroid plexus and in the peripheral and autonomic nervous systems. We extracted the amyloid fibrils from kidneys of these mice with a human mutant TTR gene and analyzed them immunochemically and electronmicroscopically. Deposited amyloid was shown to be composed of human mutant TTR and mouse serum amyloid P component. Amyloid fibril from transgenic mice was morphologically and immunohistochemically similar to that of human FAP. The most striking pathologic feature of the transgenic mice was the absence of amyloid deposition in the peripheral and autonomic nervous tissues. Thus, other intrinsic factors may be involved in amyloid deposition in the nervous tissues of human FAP.     

Change in growth kinetics of hybridoma cells entrapped in collagen gel affected by alkaline supply

The growth yields for glucose and glutamine of murine hybridoma cells entrapped in collagen gel particles were examined during the growth phase. The immobilized hybridoma cells were cultivated in a fluidized bed fermenter where the medium was circulating to supply oxygen separately. Procedures to supply an alkaline solution for adjusting the pH level strongly affected the growth yields. A direct supply of the alkaline solution to the cultivation system reduced both the growth yields for glucose and glutamine, probably due to a local increase in pH level. On the other hand, when fresh medium in which the pH was adjusted to around 8.5 was added to the cultivation system, the growth yields were unchanged even at the same pH level as when direct alkaline supply was used. These results suggest that an indirect alkaline supply could be recommended to ajust the pH level when using medium-circulating-fermenters.     

Automated monitoring of cell concentration and viability using an image analysis system

In order to automate measurements of cell concentration and viability in a suspended animal cell culture, we have developed anin situ microscopic image analysis system with an effective cell recognition algorithm. With a small amount of sample, this system can measure the cell density rapidly and aseptically. In addition, it can measure a cell size histogram including cell debris small particle distribution. These small particles have been found to be related to the viability of the mouse-mouse hybridoma STK1 cell line. By using cell debris small particle density as an indicator of cell viability, the developed system provides non-destructive viability monitoring without trypan blue staining.     

Cloning and characterization of the glutamate 1-semialdehyde aminomutase gene from Xanthomonas campestris pv. phaseoli

The gene from Xanthomonas campestris pv. phaseoli for glutamate 1-semialdehyde (GSA) aminomutase, which is involved in the C5 pathway for synthesis of -aminolevulinic acid (ALA), was cloned onto a multicopy plasmid, pUC18, by the complementation of an ALA-deficient mutant (hemL) of Escherichia coli. Subcloning of deletion fragments from the initial 3.5-kb chromosomal fragment allowed the isolation of a 1.7-kb fragment which could complement the hemL mutation. Nucleotide sequence analysis of the 1.7-kb DNA fragment revealed an open reading frame (ORF) that is located downstream from a potential promoter sequence and a ribosome-binding site. The ORF encodes a polypeptide of 429 amino acid residues, and the deduced molecular mass of this polypeptide is 45,043 Da. The amino acid sequence shows a high degree of homology to the HemL proteins from other organisms, and a putative binding site for pyridoxal 5-phosphate is conserved. Correspondence to: Y. Murooka     

Regional changes in muscle mass and strength following 20 days of bed rest, and the effects on orthostatic tolerance capacity in young subjects

To this day, many studies have suggested that prolonged bed rest (BR) affects on muscle mass and strength not only in gravity muscles but also in ungravity muscles. However, it is still unclear whether the decrease in regional muscle strength after BR is due to the alterations in the corresponding muscle mass, or not. On the other hand, if BR decreases the mass of antigravity muscles (UGM) as well as muscle strength and then increases tissue compliance of the antigravity muscles, orthostatic tolerance capacity will be decreased by the reduction in cardiac output (CO) in spite of the increase in myocardial contractility because the more decrease in venous return due to the more increase in blood pooling within the compliant tissues of the lower body. However, this is also unclear. To make these questions clear, the present study investigated the regional muscle mass and strength and orthostatic tolerance capacity before and after 20 days of bed rest in young subjects.     

Sporocarp ontogeny in Panus (Basidiomycotina): evolution and classification

Ontogenies of cultured Panus conchatus, P. rudis, and P. fulvus sporocarps were observed macroscopically and with scanning electron microscopy. Hymenophore differentiation in Panus involves periclinal growth of context hyphae below a closed surface palisade of hymenial elements, resulting in a cantharelloid appearance and radiate trama. This pattern is qualitatively different from that in Lentinus s. str., which suggests that lamellae of Panus and Lentinus are not homologous. Panus conchatus and P. rudis sporocarps have short stipes, develop directly from the mycelium, and mature in 5–10 d. Panus fulvus sporocarps have an elongate stipe, develop from a pseudosclerotium, and mature in about 3 wk, the first approximately 15 d of which involve apical elongation of a stipelike primordium that is able to dedifferentiate and regenerate cut apices. Panus conchatus and P. rudis sporocarps lacked regeneration ability. Panus conchatus sporocarps developed an ephemeral partial veil that was obliterated during sporocarp expansion. Outgroup comparison suggests that evolutionary changes in developmental programs in Panus have included: 1) delay in offset of primordium growth, with a corresponding increase in primordium size and time to maturation (hypermorphosis); 2) insertion of the pseudosclerotial stage in ontogeny; 3) gain of ability for dedifferentiation and regeneration; and 4) nonterminal gain or loss of veil tissue.     

Stable transformation of cultured cells of the liverwortMarchantia polymorpha by particle bombardment

Suspension-cultured cells (A-18 line) of the liverwortMarchanta polymorpha were bombarded by a pneumatic particle gun with plasmid pCH harbouring the hygromycin phosphotransferase (HPT) gene (hpt) under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and the nopaline synthase polyadenylation region. Nine weeks after bombardments, 128 hygromycin-resistant calluses were obtained from an approximate total of 7×10⁶ cells. Ten cell lines chosen randomly were analysed further. Southern blot analysis showed that all of the ten lines contain thehpt gene in the genome, demonstrating that these lines are transformants. An HPT enzyme activity assay confirmed the expression of the gene in all of the transformant lines.