Polymorphisms in the α-amy1 gene of wild and cultivated barley revealed by the polymerase chain reaction

-Amylases are the key enzymes involved in the hydrolysis of starch in plants. The polymerase chain reaction (PCR) was used to detect polymorphisms in the length of amplified sequences between the annealing sites of two primers derived from published -amy1 gene sequences in barley. These two primers (Bsw1 and Bsw7), flanking the promoter region and the first exon, amplified two PCR fragments in barley. One of the amplified products, with the expected length of 820 bp, appeared together with another shorter PCR band of around 750 bp. This 750-bp fragment seems to be derived from an -amylase gene not reported previously. Both of the PCR products could be amplified from the two-rowed barley varieties tested, including cv Himalaya from which the sequence information was obtained. Five of the six-rowed barley varieties also have the two PCR fragments whereas another two have only the long fragment. These two fragments seem to be unique to barley, neither of them could be amplified from other cereals; for example, wheat, rye or sorghum. These two -amylase fragments were mapped to the long arm of 6H, the location of the -amy1 genes, using wheat-barley addition lines. Amplification of genomic DNA from wild barley accessions with primers Bsw1 and Bsw7 indicated that both of the fragments could be present, or the long and short fragments could be present alone. The results also demonstrated that the genes specifying these two fragments could be independent from each other in barley. The conserved banding pattern of these two fragments in the two-rowed barley varieties implies that artificial selection from these genes may have played an important role in the evolution of cultivated barley from wild barley.     

Association of a 76 kDa polypeptide with soluble starch synthase I activity in maize (cv B73) endosperm

Soluble starch synthase (SSS) I was purified 361-fold from hand-dissected endosperm tissue of inbred maize (Zea mays, cv. B73) to specific activities ranging between 5 and 9 µmol min⁻¹ mg⁻¹. A key to this purification protocol was the introduction of a size-exclusion chromatography step, a size-based fractionation which provided abundant levels of desalted SSS forms I and II. The native molecular masses calculated for SSS forms I and II were 75.5 kDa and 180 kDa, respectively. SSSI was then further purified by hydrophobic interaction chromatography on Phenyl-Superose and by FPLC on Mono Q. Analysis of column peaks by SDS—PAGE and scanning densitometry revealed that a 76 kDa polypeptide is strongly correlated with SSSI activity. Antibodies were then generated against a 76 kDa polypeptide extracted from starch granules. These antibodies, which were monospecific for the soluble 76 kDa polypeptide, neutralized greater than 90% of SSSI activity, and precipitated the 76 kDa protein. These results establish the 76 kDa protein as an SSSI in the B73 line of inbred maize. An immunologically similar 76 kDa protein also appears to be tightly associated with the starch granule.     

The promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice)

C₄ plants have two carboxylases which function in photosynthesis. One, phosphoenolpyruvate carboxylase (PEPC) is localized in mesophyll cells, and the other, ribulose bisphosphate carboxylase (RuBPC) is found in bundle sheath cells. In contrast, C₃ plants have only one photosynthetic carboxylase, RuBPC, which is localized in mesophyll cells. The expression of PEPC in C₃ mesophyll cells is quite low relative to PEPC expression in C₄ mesophyll cells. Two chimeric genes have been constructed consisting of the structural gene encoding β-glucuronidase (GUS) controlled by two promoters from C₄ (maize) photosynthetic genes: (i) the PEPC gene (pepc) and (ii) the small subunit of RuBPC (rbcS). These constructs were introduced into a C₃ cereal, rice. Both chimeric genes were expressed almost exclusively in mesophyll cells in the leaf blades and leaf sheaths at high levels, and no or very little activity was observed in other cells. The expression of both genes was also regulated by light. These observations indicate that the regulation systems which direct cell-specific and light-inducible expression of pepc and rbcS in C₄ plants are also present in C₃ plants. Nevertheless, expression of endogenous pepc in C₃ plants is very low in C₃ mesophyll cells, and the cell specificity of rbcS expression in C₃ plants differs from that in C₄ plants. Rice nuclear extracts were assayed for DNA-binding protein(s) which interact with a cis-regulatory element in the pepc promoter. Gel-retardation assays indicate that a nuclear protein with similar DNA-binding specificity to a maize nuclear protein is present in rice. The possibility that differences in pepc expression in a C₃ plant (rice) and C₄ plant (maize) may be the result of changes in cis-acting elements between pepc in rice and maize is discussed. It also appears that differences in the cellular localization of rbcS expression are probably due to changes in a trans-acting factor(s) required for rbcS expression.     

Overview of a quality assurance/quality control compliance program consistent with FDA regulations and policies for somatic cell and gene therapies: a four year experience

Somatic cell and gene therapy involve the application of biological technologies to an individual patient through the use of living cells which provide a therapeutic benefit (Aliski, 1991). Various forms of cellular and gene therapies are being developed and evaluated in an increasing number of clinical trials for congential and acquired disorders. The potential and progress of these therapeutic applications have resulted in an increasing effort by the Food and Drug Administration (FDA) to develop the regulatory framework under which these therapeutic approaches would insure safety and efficacy, the primary mandate of the FDA.Over five years ago Cellcor began to define the parameters, specifications, and conditions relevant to a Quality Assurance/Quality Control (QA/QC) program that has evolved to insure safety and maximize the efficacy of applications of the company'sex vivo technology, autolymphocyte therapy. Autolymphocyte therapy is an outpatient form of somatic cell immunotherapy based upon the infusion of T cells that have been activatedex vivo using a combination of previously generated autologous cytokines and an anti-CD3 monoclonal antibody.We have been able to demonstrate the feasibility for the safe, controlled, and consistent preparation and delivery of a cellular therapy by application of relevant GMP regulations. This presentation reviews aspects of this program and chronicles our experience which at present amounts to over 4400 infusions for over 700 patients. This program provides a high degree of assurance that a cellular therapy program can be carried out in a multisite mode involving hundreds of patients through the strict adherence to cGMP as set forth in existing regulations. It would be prudent that developers of cellular andex vivo gene therapies establish a similar cell processing and QA/QC infrastructure at an early developmental stage to optimize safety and reproducibility and facilitate regulatory review.     

Cell cycle dependence of cytotoxicity and clastogenicity induced by treatment of synchronized human diploid fibroblasts with sodium fluoride

To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures. (1) The cells were synchronized at G₀/G₁ phase by a period of growth in medium containing 1% serum (low serum medium). (2) The cells were synchronized at the G₁/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G₁ phase or G₂ phase, and for each of three 3-h periods during the S phase which lasted 9 h. Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G₂ phase. Little lethality was observed in cultures in G₁ phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and / or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.     

The Presequence of a Precursor to the {delta}-Subunit of Sweet Potato Mitochondrial F1ATPase is not Sufficient for the Transport of {beta}-Glucuronidase (GUS) into Mitochondria of Tobacco, Rice and Yeast Cells

A precursor to the     

Roles of multiple glucose transporters in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, TRK1 and TRK2 are required for high- and low-affinity K+ transport. Among suppressors of the K+ transport defect in trk1 delta trk2 delta cells, we have identified members of the sugar transporter gene superfamily. One suppressor encodes the previously identified glucose transporter HXT1, and another encodes a new member of this family, HXT3. The inferred amino acid sequence of HXT3 is 87% identical to that of HXT1, 64% identical to that of HXT2, and 32% identical to that of SNF3. Like HXT1 and HXT2, overexpression of HXT3 in snf3 delta cells confers growth on low-glucose or raffinose media. The function of another new member of the HXT superfamily, HXT4 (previously identified by its ability to suppress the snf3 delta phenotype; L. Bisson, personal communication), was revealed in experiments that deleted all possible combinations of the five members of the glucose transporter gene family. Neither SNF3, HXT1, HXT2, HXT3, nor HXT4 is essential for viability. snf3 delta hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells are unable to grow on media containing high concentrations of glucose (5%) but can grow on low-glucose (0.5%) media, revealing the presence of a sixth transporter that is itself glucose repressible. This transporter may be negatively regulated by SNF3 since expression of SNF3 abolishes growth of hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells on low-glucose medium. HXT1, HXT2, HXT3, and HXT4 can function independently: expression of any one of these genes is sufficient to confer growth on medium containing at least 1% glucose. A synergistic relationship between SNF3 and each of the HXT genes is suggested by the observation that SNF2 hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells and snf3 delta HXT1 HXT2 HXT3 HXT4 cells are unable to grow on raffinose (low fructose) yet SNF3 in combination with any single HXT gene is sufficient for growth on raffinose. HXT1 and HXT3 are differentially regulated. HXT1::lacZ is maximally expressed during exponential growth whereas HXT3::lacZ is maximally expressed after entry into stationary phase.     

Chemical synthesis of insulin-like peptide 1 and its potential role in vitellogenesis of the kuruma prawn Marsupenaeus japonicus

The insulin superfamily comprises a group of peptides with diverse physiological functions and is conserved across the animal kingdom. Insulin-like peptides (ILPs) of crustaceans are classified into four major types: insulin, relaxin, gonadulin, and androgenic gland hormone (AGH)/insulin-like androgenic gland factor (IAG). Of these, the physiological functions of AGH/IAG have been clarified to be the regulation of male sex differentiation, but those of the other types have not been uncovered. In this study, we chemically synthesized Maj-ILP1, an ILP identified in the ovary of the kuruma prawn Marsupenaeus japonicus, using a combination of solid-phase peptide synthesis and regioselective disulfide bond formation reactions. As the circular dichroism spectral pattern of synthetic Maj-ILP1 is typical of other ILPs reported thus far, the synthetic peptide likely possessed the proper conformation. Functional analysis using ex vivo tissue incubation revealed that Maj-ILP1 significantly increased the expression of the yolk protein genes Maj-Vg1 and Maj-Vg2 in the hepatopancreas and Maj-Vg1 in the ovary of adolescent prawns. This is the first report on the synthesis of a crustacean ILP other than IAGs and also shows the positive relationship between the reproductive process and female-dominant ILP.