Plant regeneration from sorghum anther cultures and field evaluation of progeny

Summary Embryogenic calli were obtained from a hybrid line of Sorghum bicolor (L.) Moench anthers with mid to late uninucleate pollen cultured in N₆ medium supplemented with 3.0% sucrose, 2.0 mg/1 2,4-D, 0.8% agar and incubated at 30°C, which was the optimum temperature. The regeneration of embryos was obtained from the embryogenic calli cultured in modified MS medium supplemented with 3.0% sucrose, 2.0 mg/1 BAP combined with 0.3 mg/1 IAA and 0.8% agar. A total of 248 doubled haploids and 12 haploid plants were regenerated. In a subsequent field study, the selfed progeny from anther culture (designated as the anther culture-2, [A₂] generation) derived families was compared with both the F₂ and the F₁ for agronomic and morphological traits. Significant differences were noticed between the family means of both A₂ and F₂ for all the quantitative traits studied. The distinctive difference in the behavior of the A₂ families in comparison with the F₂ was established by within family variance, which was significant in F₂ for days to 50 per cent flowering, plant height, panicle length, leaf area index, dry matter production, harvest index and grain yield and was non-significant in A₂. Male sterility, one of the potentially important traits, currently exploited in the hybrid seed production of cereals, including CSH5 hybrid sorghum and the morphological traits (panicle shape, compactness, grain color, glume color and nature of the leaf sheath) segregated in the F₂. Such segregation was not observed within A₂ families and they bred true to their respective A₁ plants, indicating the rapid attainment of homozygosity/uniformity. The present study establishes the gametophytic origin of anther culture derived families and indicates the possibility of rapid production of homozygous lines which can be used as recombinant inbreds.Abbreviations N₆ Chu et al. (1975) - MS Murashige and Skoog (1962) - 2,4-D, 2 4-Dichlorophenoxyacetic acid - IAA Indole-3 -acetic acid - BAP Benzylaminopurine - A₁ regenerated plants - A2 selfed progeny from A₁     

A comparison of mutation induction in diploid and tetraploid rice

Summary A comparative assessment of the frequency and spectrum of chlorophyll mutations in the M ₂ and M ₃ of three diploids and one tetraploid of rice after X-irradiation was made. As well as a linear relationship of the frequency of mutations with the dose of mutagen, a saturation effect was also evident. Among the three diploids, the maximum frequency of mutations was observed in T. N. 1, followed by G. E. B. 24 and A. S. D. 8. The diploid of G. E. B. 24 showed a higher frequency of mutations than the tetraploid when measured on the M₁ plant basis only. The diploids showed a higher rate of mutations in the M ₂ than in the M₃.There was no relationship between the frequency of different types of chlorophyll mutations and the dose of X-rays. Albina, occurred in greater proportion than the other types of mutations in the diploids as well as in the tetraploid. The diploid showed a wider spectrum in the M ₂, whereas the tetraploid recorded the maximum types of mutations in the M ₃. Striking differences in the spectrum and the relative frequency of each type were observed among the three diploids and also between the diploid G. E. B. 24 and its tetraploid.The frequency and spectrum of induced mutations in the diploid and autotetraploid provided an insight into the genetic behaviour of the diploid and autotetraploid of G. E. B. 24, indicating that the genetics of diploidisation of the existing diploid rice may give evidence on the nature of the evolutionary pathway.     

Non‐specific immunity and disease resistance are enhanced by the polysaccharide fraction of a marine chlorophycean macroalga in Oreochromis niloticus (Linnaeus, 1758)

Polysaccharides (PF) from marine macroalgae, Caulerpa scalpelliformis were extracted and tested for its potential immunostimulatory and disease resistance properties in fish. Five groups of Nile tilapia (n = 6), Oreochromis niloticus (Linnaeus, 1758) were intraperitoneally administered with the different doses of PF (2, 20 or 200 mg/kg body weight) or with yeast‐derived commercial immunostimulant, Macrogard^? (20 mg/kg body weight), to compare the effectiveness. An untreated control group was also maintained. A total of fifteen fibre reinforced plastic tanks (150 L, ambient temperature and light conditions) were used, with triplicate tanks for each group. Only four fish per tank (totally 12 fish from a group) were taken at random and assayed. PF enhanced all the tested non‐specific serum immune responses namely lysozyme, myeloperoxidase, antiprotease, and bactericidal activities. There was an upregulation of the genes encoding IL‐1β, lysozyme and TNF‐α in the spleen of PF injected fish as compared to the control group. In order to study the overall functional immunity, disease resistance test was conducted. Another five groups of fish (n = 10) were treated by intraperitoneal injection with different doses of PF or Macrogard^? or untreated as mentioned earlier in triplicates (30 fish per group in three tanks, totally 150 fish in 15 tanks). Seven days post treatment, fish were challenged by intraperitoneal administration of live virulent Aeromonas hydrophila. PF treated fish were protected with significant reduction in the mortality and the consequent increased relative percent survival (RPS) of 92 in the least (2 mg/kg) and middle dose (20 mg/kg) groups. The disease resistance experiment was repeated again but this time, fish were challenged 21 days post treatment that resulted in RPS of 50 for the middle dose. The results clearly show that the intraperitoneal administration of the polysaccharide fraction had a stimulating effect on the non‐specific immune responses, immune gene expression and disease resistance.     

Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins

As part of an effort to understand how proteins are imported into the peroxisome, we have sought to identify the peroxisomal targeting signals in four unrelated peroxisomal proteins: human catalase, rat hydratase:dehydrogenase, pig D-amino acid oxidase, and rat acyl-CoA oxidase. Using gene fusion experiments, we have identified a region of each protein that can direct heterologous proteins to peroxisomes. In each case, the peroxisomal targeting signal is contained at or near the carboxy terminus of the protein. For catalase, the peroxisomal targeting signal is located within the COOH-terminal 27 amino acids of the protein. For hydratase:dehydrogenase, D-amino acid oxidase, and acyl-CoA oxidase, the targeting signals are located within the carboxy-terminal 15, 14, and 15 amino acids, respectively. A tripeptide of the sequence Ser-Lys/His-Leu is present in each of these targeting signals as well as in the peroxisomal targeting signal identified in firefly luciferase (Gould, S.J., G.-A. Keller, and S. Subramani. 1987. J. Cell Biol. 105:2923-2931). When the peroxisomal targeting signal of the hydratase:dehydrogenase is mutated so that the Ser-Lys-Leu tripeptide is converted to Ser-Asn-Leu, it can no longer direct proteins to peroxisomes. We suggest that this tripeptide is an essential element of at least one class of peroxisomal targeting signals.     

The carboxyl-terminal tripeptide serine-lysine-leucine of firefly luciferase is necessary but not sufficient for peroxisomal import in yeast.

Firefly luciferase is imported into peroxisomes in insects, mammals, plants, and yeast, which implies that the mechanism of protein translocation into peroxisomes has been conserved during eukaryotic evolution. The carboxyl-terminal tripeptide serine-lysine-leucine in luciferase acts as a peroxisomal import signal in mammalian cells. We have investigated whether this tripeptide is also involved in translocation of firefly luciferase into peroxisomes in yeast (Saccharomyces cerevisiae). We show by gene fusion experiments that the carboxyl-terminal 104 amino acids of luciferase can direct a heterologous protein to yeast peroxisomes. Luciferase mutant proteins were tested for their ability to be imported into yeast peroxisomes in vivo. We demonstrate that mutations in the carboxyl-terminal serine-lysine-leucine tripeptide abolish translocation of the protein into yeast peroxisomes. However, when a passenger protein was tagged at its carboxyl terminus with this tripeptide the fusion protein did not go to peroxisomes. These results indicate that, in yeast, the tripeptide is necessary but not sufficient for peroxisomal import.     

Overexpression of Pex15p, a phosphorylated peroxisomal integral membrane protein required for peroxisome assembly in S.cerevisiae, causes proliferation of the endoplasmic reticulum membrane.

We have cloned PEX15 which is required for peroxisome biogenesis in Saccharomyces cerevisiae. pex15Delta cells are characterized by the cytosolic accumulation of peroxisomal matrix proteins containing a PTS1 or PTS2 import signal, whereas peroxisomal membrane proteins are present in peroxisomal remnants. PEX15 encodes a phosphorylated, integral peroxisomal membrane protein (Pex15p). Using multiple in vivo methods to determine the topology, Pex15p was found to be a tail-anchored type II (Ncyt-Clumen) peroxisomal membrane protein with a single transmembrane domain near its carboxy-terminus. Overexpression of Pex15p resulted in impaired peroxisome assembly, and caused profound proliferation of the endoplasmic reticulum (ER) membrane. The lumenal carboxy-terminal tail of Pex15p protrudes into the lumen of these ER membranes, as demonstrated by its O-glycosylation. Accumulation in the ER was also observed at an endogenous expression level when Pex15p was fused to the N-terminus of mature invertase. This resulted in core N-glycosylation of the hybrid protein. The lumenal C-terminal tail of Pex15p is essential for targeting to the peroxisomal membrane. Furthermore, the peroxisomal membrane targeting signal of Pex15p overlaps with an ER targeting signal on this protein. These results indicate that Pex15p may be targeted to peroxisomes via the ER, or to both organelles.     

Functional Mapping of the DNA Binding Domain of Bovine Papillomavirus E1 Protein

Bovine papillomavirus type 1 (BPV-1) requires viral proteins E1 and E2 for efficient DNA replication in host cells. E1 functions at the BPV origin as an ATP-dependent helicase during replication initiation. Previously, we used alanine mutagenesis to identify two hydrophilic regions of the E1 DNA binding domain (E1DBD), HR1 (E1(179-191)) and HR3 (E1(241-252)), which are critical for sequence-specific recognition of the papillomavirus origin. Based on sequence and structure, these regions are similar in spacing and location to DNA binding regions A and B2 of T antigen, the DNA replication initiator of simian virus 40 (SV40). HR1 and A are both part of extended loops which are supported by residues from the HR3 and B2 alpha-helices. Both elements contain basic residues which may contact DNA, although lack of cocrystal structures for both E1 and T antigen make this uncertain. To better understand how E1 interacts with origin DNA, we used random mutagenesis and a yeast one-hybrid screen to select mutations of the E1DBD which disrupt sequence-specific DNA interactions. From the screen we selected seven single point mutants and one double point mutant (F175S, N184Y/K288R, D185G, V193M, F237L, K241E, R243K, and V246D) for in vitro analysis. All mutants tested in electrophoretic mobility shift assays displayed reduced sequence-specific DNA binding compared to the wild-type E1DBD. Mutants D185G, F237L, and R243K were rescued in vitro for DNA binding by the replication enhancer protein E2. We also tested the eight mutations in full-length E1 for the ability to support DNA replication in Chinese hamster ovary cells. Only mutants D185G, F237L, and R243K supported significant DNA replication in vivo which highlights the importance of E1DBD-E2 interactions for papillomavirus DNA replication. Based on the specific point mutations examined, we also assigned putative roles to individual residues in DNA binding. Finally, we discuss sequence and spacing similarities between E1 HR1 and HR3 and short regions of two other DNA tumor virus origin-binding proteins, SV40 T antigen and Epstein-Barr virus nuclear antigen 1 (EBNA1). We propose that all three proteins use a similar DNA recognition mechanism consisting of a loop structure which makes base-specific contacts (HR1) and a helix which primarily contacts the DNA backbone (HR3).     

Estimation of Several Missing Values in F-Square Designs

The present study deals with the estimation of several missing values in F-square designs. The estimating equations for the non-iterative least squares estimation of Missing Values and explicit expressions for the estimators of the particular patterns of Missing Values are presented. This procedure is illustrated with the help of a numerical example.