Non-allelic interaction conditioning spikelet sterility in an F2 population of indica/japonica cross in rice

Significant segregation of spikelet fertility occurred in an F₂ population derived from a spikelet fertility-normal F₁ hybrid produced by a cross between Palawan, a japonica variety, and IR42, an indica variety. To identify factors controlling the fertility segregation, we used 104 RFLP markers covering all 12 rice chromosomes to investigate the association of spikelet fertility and marker segregation. We found that the segregation of two sets of gene pairs was significantly (P < 0.001) associated with fertility segregation. The first pair of genes was linked to RFLP marker RG778 on chromosome 12 and RFLP markers RG690/RG369 on chromosome 1. A significant reduction in fertility was observed when the plants were homozygote at RG778 with the indica allele as well as homozygote at RG690/RG369 with the japonica allele. The second pair of genes was linked to RG218 on chromosome 12 and RG650 on chromosome 7, respectively. The recombinant homozygote at these two loci showed a significant reduction on spikelet fertility. The non-allelic interaction effect was further modified by a gene linked to RG778, resulting in even lower fertility. The results of this study provides the first evidence of chromosomal localization of sporophytic sterility genes whose interaction can result in a reduction of spikelet fertility in the F₂ derived from fertility-normal F₁.     

The distribution of Gossypium hirsutum chromatin in G. barbadense germ plasm: molecular analysis of introgressive plant breeding

Cotton is unusual among major crop plants in that two cross-fertile species are widely cultivated for a common economic product, fiber. Both historical evidence and classical genetic studies suggest that many improved forms of Gossypium barbadense (Sea Island, Egyptian, and Pima cottons) may include chromatin derived from G. hirsutum. Using 106 restriction fragment length polymorphism (RFLP) loci well distributed across the cotton genome, we revealed the amount and genomic distribution of G. hirsutum chromatin in 54 G. barbadense collections from around the world. The average G. barbadense collection was comprised of 8.9% alleles apparently derived from G. hirsutum. Pima cultivars (7.3 %) had fewer G. hirsutum alleles than Sea Island (9.0%) or Egyptian (9.6%) cultivars. G. hirsutum alleles were not randomly distributed, as 57.5% of the total introgression observed was accounted for by five specific chromosomal regions that span less than 10% of the genome. The average length of an introgressed chromosome segment was 12.9 cM. Overlap of introgressed chromatin in different breeding programs hints that retention of these G. hirsutum chromosomal segments may impart a selective advantage to G. barbadense genotypes. Although cluster analysis generally grouped germ plasm from common classes and/or breeding programs together, no 2 genotypes were identical — thus differences in the length and repertoire of introgressed chromosome segments also permit DNA fingerprinting of G. barbadense cultivars.     

PSEUDO-NITZSCHIA PUNGENS AND P. MULTISENES (BACILLARIOPHYCEAE): NUCLEAR RIBOSOMAL DNAS AND SPECIES DIFFERENCES1

The region of the nuclear ribosomal DNA (rDNA) operon containing the small subunit (SSU), internal transcribed spacer 1 (ITS1), and a portion of the 5.8s rDNA gene was sequenced in one isolate each of Pseudo-nitzschia multiseries (Hasle) Hasle and Pseudo-nitzschia pungens (Grunow in Cleve & Möller) Hasle. The SSUs of these two species were highly similar, differing only in 14 point mutations and one insertion/deletion in 1774 bp. The ITS1 sequences were more variable, with 57 point mutations and three insertion/deletions in 257 bp. There were no differences in 44 bp of the 5.8S sequences. Restriction fragment patterns (RFPs) for the restriction endonucleases HaeIII, Hha1, and Rsa1 for 13 isolates of P. multiseries from the Atlantic, Pacific, and Gulf coasts of the United States and 16 isolates of P. pungens from the three coasts of the United States, in addition to Japan and China, were compared. There were differences between the RFPs of P. multiseries and P. pungens that corresponded to sites mapped by the DNA sequences, but no infraspecific variation in RFPs was observed for either species. The differences in RFPs correlate with morphological, immunological, and other rDNA differences and support the recognition of these taxa as separate species.     

Effect of DNA fragment size on transformation frequencies in tobacco (Nicotiana tabacum) and maize (Zea mays)

During eukaryotic cell transformation, the transforming DNA must enter the host cell, traverse the cytoplasm and enter the nucleus before becoming stably integrated into the genome. The limiting step for plant protoplast transformation may lie at the cell membrane, the nuclear membrane, or at the integration step. We show here that the size of the DNA fragment containing the selectable marker used to monitor transformation can directly affect the efficiency of stable transformation. In both tobacco and maize protoplasts, the smallest DNA fragments gave the highest stable transformation frequencies.     

Prothrombin cleavage by human vascular smooth muscle cells: A potential alternative pathway to the coagulation cascade

Thrombin is a potent mitogen for human vascular smooth muscle cells (HVSMC) and its enzymatic activity is required for this function. The present study demonstrates that prothrombin is also mitogenic for HVSMC due to the generation of enzymatically active thrombin which occurs upon incubation of prothrombin with the cells. Analysis by SDS-PAGE, immunoblotting, and amino acid sequencing revealed that prothrombin incubated with HVSMC undergoes limited proteolysis. Prethrombin 1 was formed through cleavage at R¹⁵⁵-S¹⁵⁶. Cleavage at R²⁷¹-T²⁷² generated fragment 1.2 and prethrombin 2 whilst cleavage at R²⁸⁴-T²⁸⁵ yielded truncated prothrombin 2 (prethrombin 2′). However, cleavage at R³²⁰-I³²¹ which, during prothrombin activation produces two-chain α-thrombin, was not detectable. Studies on HVSMC-conditioned medium revealed that a similar pattern of prothrombin cleavage occurred by a cell-secreted factor(s). Amidolytic activity analysis indicated that 1–3% catalytically active thrombin-like activity was generated upon incubation of prothrombin with HVSMC-conditioned medium. By treating conditioned medium with various classes of proteinase inhibitors or hirudin, it was determined that prothrombin is cleaved by a cell-derived serine proteinase-like factor(s) at R²⁷¹-S²⁷² and by α-thrombin at R¹⁵⁵-S¹⁵⁶ and R²⁸⁴-T²⁸⁵. Antibodies neutralising the activity of either urokinase, tissue plasminogen activator, or factor Xa failed to alter the prothrombin cleaving activity of conditioned medium. This activity which may catalyse an alternative pathway for the generation of thrombin, was eluted from a gel filtration column as a single peak with apparent molecular mass of 30–40 kDa. © 1995 Wiley-Liss, Inc.     

Mammalian expression of single chain variable region fragments dimerized by Fc regions

To promote application of a single chain variable region fragment (sFv) in immunoglobulins, a sFv gene was connected to an IgG1 Fc gene, designated as a sFvc gene, and used for transfection of Sp2/0. As a result, the sFvc protein was found to be secreted in a dimeric form. It is thus felt that the sFvc protein, which mimicks the shape of a naturally occurring antibody, can be simple and useful to reproduce divalency and Fc-associated effecter functions as seen in a natural antibody.Abbreviations Abbreviations sFv single chain variable region fragment - Fc constant region of immunoglobulin - sFvc single chain variable region fragment with an Fc region     

Plasmid RFLP profiling and DNA homology inThermus isolated from hot springs of different geographical areas

Thirty-four strains belonging to various species of the genus Thermus (T. aquaticus, "T. thermophilus," "T. brockianus," T. scotoductus, and genomic species 2) isolated from hot springs of different geographical areas were examined for plasmid content and restriction fragment length polymorphism (RFLP) of plasmid DNAs. The four strains of the numerical taxonomy cluster E of genomic species 2 did not harbor plasmid DNA. Overall examination of the HindIII-RFLP profiling of plasmid DNA showed considerable variability between and within genomic species, with the exception of presumed clonal isolates. In spite of this heterogeneity, HindIII plasmid digests within a numerical taxonomic cluster gave a subset of restriction fragments of similar or identical length. Strains belonging to genomic species 2 or unclassified isolates from S. Pedro do Sul that harbored plasmid DNA (7 of the 14 strains studied) exhibited strong DNA homology between plasmid regions. No homologous sequences to these plasmid regions were found in chromosomal DNA from strains isolated from S. Pedro do Sul in which no plasmids were detected. The strains belonging to T. scotoductus formed two plasmid DNA homology groups, as estimated by probing with a plasmid fragment that coincided with the two numerical taxonomy clusters proposed previously. Among the other species, homology of plasmid regions was also found between some strains. Strong homology was also found between plasmid regions from some strains of different taxonomic groups, isolated from the same and from different sources, suggesting that these sequences are highly conserved in plasmids present in Thermus. For plasmid-containing strains, results of plasmid RFLP profiling/DNA homology appear promising for the typing of Thermus at the level of biotypes or of individual strains, namely, for monitoring the diversity and frequency of isolates from a particular hot spring. Received: 24 October 1994 / Accepted: 6 March 1995     

Isolation and characterization of alpha-endorphin and gamma-endorphin from single human pituitary glands

alpha-Endorphin and gamma-endorphin, two closely related peptides of the pro-opiomelanocortin family with characteristic biological activities, were purified to homogeneity from single human pituitary glands and chemically identified. Isolation of the peptides was based on size fractionation by Sephadex G-75 chromatography followed by two HPLC steps using reverse-phase and paired-ion reverse-phase systems and was monitored by radioimmunoassay. During the isolation procedure alpha- and gamma-endorphin-sized material behaved chromatographically and immunologically indistinguishably from synthetic alpha- and gamma-endorphin. The amino acid composition and NH2-terminus of isolated peptides demonstrated their identity as authentic alpha-endorphin and gamma-endorphin. Acetylated forms were absent. In addition, evidence is provided that large forms with alpha- and gamma-endorphin immunoreactivity detected during gel filtration are human lipotropin-(1-74) and -(1-75), respectively. The data substantiate that alpha-endorphin and gamma-endorphin exist as endogenous peptides in the human pituitary gland.