The use of bulk segregant analysis to identify a RAPD marker linked to leaf rust resistance in barley

An F₂ population from a cross between barley accession Q21861 and the Australian barley variety Galleon was used to develop RAPD markers for resistance to barley leaf rust (Puccinia hordei). Resistant and susceptible DNA bulks were constructed following the classification of F₂ plants by leaf rust infection type. Bulked segregant analysis was then used to identify a 2.7-kb marker, designated OU02₂₇₀₀ and located approximately 12cM from RphQ, a leaf rust resistance gene in Q21861. The marker was generated by PCR with the oligonucleotide primer OPU-02 (Operon). Infection types of F₃ progeny were used to confirm assignment of F₂ genotypes. OU02₂₇₀₀ was shown, retrospectively, to be useful in the identification of individual F₂ plants that had been originally misclassified as having susceptible infection types. Both the RAPD marker and RphQ will be potentially useful in the development of new barley cultivars.     

NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5.

The double-stranded RNA binding domain (dsRBD) is an approximately 65 amino acid motif that is found in a variety of proteins that interact with double-stranded (ds) RNA, such as Escherichia coli RNase III and the dsRNA-dependent kinase, PKR. Drosophila staufen protein contains five copies of this motif, and the third of these binds dsRNA in vitro. Using multinuclear/multidimensional NMR methods, we have determined that staufen dsRBD3 forms a compact protein domain with an alpha-beta-beta-beta-alpha structure in which the two alpha-helices lie on one face of a three-stranded anti-parallel beta-sheet. This structure is very similar to that of the N-terminal domain of a prokaryotic ribosomal protein S5. Furthermore, the consensus derived from all known S5p family sequences shares several conserved residues with the dsRBD consensus sequence, indicating that the two domains share a common evolutionary origin. Using in vitro mutagenesis, we have identified several surface residues which are important for the RNA binding of the dsRBD, and these all lie on the same side of the domain. Two residues that are essential for RNA binding, F32 and K50, are also conserved in the S5 protein family, suggesting that the two domains interact with RNA in a similar way.     

Identification and characterization of Uss1p (Sdb23p): a novel U6 snRNA-associated protein with significant similarity to core proteins of small nuclear ribonucleoproteins.

The SDB23 gene of Saccharomyces cerevisiae was isolated in a search for high copy-number suppressors of mutations in a cell cycle gene, DBF2, SDB23 encodes a 21,276 Da protein with significant sequence similarity to characterized mammalian snRNP core proteins. Examination of multiple sequence alignments of snRNP core proteins with Sdb23p indicates that all of these proteins share a number of highly conserved residues, and identifies a novel motif for snRNP core proteins. Sdb23p is essential for cell viability and is required for nuclear pre-mRNA splicing both in vivo and in vitro. Extracts prepared from Sdb23p-depleted cells are unable to support splicing and have vastly reduced levels of U6 snRNA. The stability of U1, U2, U4 and U5 spliceosomal snRNAs is not affected by the loss of Sdb23p. Antibodies raised against Sdb23p strongly coimmunoprecipitate free U6 snRNA and U4/U6 base-paired snRNAs. These results establish that SDB23 encodes a novel U6 snRNA-associated protein that is essential for the stability of U6 snRNA. We therefore propose the more logical name USS1 (U-Six SnRNP) for this gene.     

SOURCES OF INORGANIC CARBON FOR PHOTOSYNTHESIS BY THREE SPECIES OF MARINE DIATOM1

The utilization of inorganic carbon by three species of marine diatom, Skeletonema costatum (Grev.) Cleve. Ditylum brightwellii (West) Grun., and Chaetoceros calcitrans Paulsen was investigated using an inorganic carbon isotopic disequilibnum technique and inorganic carbon dose-response curves. Stable carbon isotope data of the diatoms are also presented. Observed rates of photosynthetic oxygen evolution were greater than could be accounted for by the theoretical rate of CO₂ supply from the uncatalyzed dehydration of HCO₃^? in the external medium, suggesting use of HCO₃^? as an inorganic carbon source. Data from the isotopic disequilibrium experiment demonstrate the use of both HCO₃^? and CO₂ for photosynthesis. Carbon isotope discrimination values support the use of HCO₃^? by the diatoms.     

Salmonella typhimurium secreted invasion determinants are homologous to Shigella lpa proteins

Salmonella typhimurium secreted proteins (Ssp) were previously implicated in epithelial cell invasion. Here we describe four genes ( sspB , sspC , sspD , and sspA ), located between spaT and prgH , which encode proteins of 63, 42, 36, and 87 kDa, respectively. These Ssp are homologous to Shigella flexneri secreted proteins lpaB, lpaC, lpaD and lpaA. A non-invasive mutant with a transposon insertion in sspC lacks Ssp of 87,42 and 36 kDa. Complementation analyses show that sspC and sspD encode the 42 and the 36 kDa Ssp, while the 87 kDa Ssp is encoded by sspA . sspC and sspD , but not sspA are required for invasion. Amino-terminal sequencing shows that SspC and SspA are secreted without amino-terminal processing. We further demonstrate that Ssp secretion requires proteins encoded by prgHIJK , homologous to the Shigella lpa secretion system, since SspA is abundantly secreted by wild-type bacteria but is completely retained within the cellular fraction of a prgHIJK mutant. A precipitate containing abundant SspC and three other major Ssp of 63,59 and 22 kDa was isolated from culture supernatants of wild-type bacteria. These data indicate that major secreted invasion determinants of S. typhimurium are structurally and functionally homolgous to S. flexneri lpa proteins.     

Thermal dependence of contractile properties of single skinned muscle fibres from Antarctic and various warm water marine fishes including Skipjack Tuna (Katsuwonus pelamis) and Kawakawa (Euthynnus affinis)

Summary Single fast fibres and small bundles of slow fibres were isolated from the trunk muscles of an Antarctic (Notothenia neglecta) and various warm water marine fishes (Blue Crevally,Carangus melampygus; Grey Mullet,Mugil cephalus; Dolphin Fish,Coryphaena hippurus; Skipjack-tuna,Katsuwonus pelamis and Kawakawa,Euthynuus affinis). Fibres were chemically skinned with the nonionic detergent Brij 58.For warm water species, maximum Ca²⁺-activated tension (P ₀) almost doubled between 5–20°C with little further increase up to 30°C. However, when measured at their normal body temperatures,P ₀ values for fast fibres were similar for all species examined, 15.7–22.5 N · cm^–2. Ca²⁺-regulation of contraction was disrupted at temperatures above 15°C in the Antarctic species, but was maintained at up to 30°C for warm water fish.Unloaded (maximum) contraction speeds (V _max) of fibres were determined by the slacktest method. In general,V _max was approximately two times higher in white than red muscles for all species studied, except Skipjack tuna. For Skipjack tuna,V _max of superficial red and white fibres was similar (15.7 muscle lengths · s^–1 (L ₀ · s^–1)) but were 6.5 times faster than theV _max of internal red muscle fibres (2.4±0.2L ₀ · s^–1) (25°C). V _max forN. neglecta fast fibres at 0–5°C (2–3L ₀ · s^–1) were similar to that of warm water species measured at 10–20°C. However, when measured at their normal muscle temperatures, theV _max for the fast muscle fibres of the warm water species were 2–3 times higher than that forN. neglecta.In general,Q _{10(15–30°C)} values forV _max were in the range 1.8–2.0 for all warm water species studied except Skipjack tuna.V _max for the internal red muscle fibres of Skipjack tuna were much more temperature dependent (Q _{10(15–30°C)}=3.1) (P<0.01) than for superficial red or white muscle fibres. The proportion of slower red muscle fibres in tuna (28% for 1 kg Skipjack) is 3–10 times higher than for most teleosts and is related to the tuna's need to sustain high cruising speeds. We suggest that the 8–10°C temperature gradient that can exist in Skipjack tuna between internal red and white muscles allows both fibre types to contract at the same speed. Therefore, in tuna, both red and white muscle may contribute to power generation during high speed swimming.     

Cytomorphology of primary small-cell (Merkel-cell) carcinoma of the skin in fine needle aspirates

The histologic appearance of primary small-cell carcinoma of the skin (the so-called Merkel-cell tumor) is similar to other small-cell tumors that may metastasize to the dermis. Significance has been placed on the electron microscopic appearance of this tumor since the ultrastructural features of this neoplasm are helpful in distinguishing it from most of the other neoplasms considered in the differential diagnosis. To determine whether any additional morphologic criteria might exist to distinguish this neoplasm, the fine needle aspirate appearance of a primary small-cell carcinoma of the skin was studied and compared to that of similar preparations of other small-cell tumors that could potentially involve the dermis. Cells of this unusual tumor were round and showed neither cohesiveness nor nuclear molding. Mitoses were numerous. The chromatin pattern was bland. The cytologic features of this tumor can aid in the distinction of primary small-cell carcinoma of the skin from other metastatic small-cell neoplastic lesions in the dermis of adults.     

Fc-receptor mediated protein phosphorylation in murine peritoneal macrophages

The effect of Fc receptor engagement on protein phosphorylation in murine peritoneal macrophages has been investigated. Treatment of macrophage cultures with insoluble immune complexes resulted in enhanced phosphorylation of six proteins at 73, 66, 53, 37, 31 and 25 kD. Comparison of the protein phosphorylation patterns induced by immune complexes with those induced by agents which mimic the actions of well known intracellular second messengers (i.e., A23187, dibutyryl cAMP, or phorbol myristate acetate) revealed substantial similarity between Fc receptor induced events and those induced in response to phorbol diesters. There were, however, two phosphorylated proteins which were only seen following stimulation with immune complexes. Thus, more than one kind of protein kinase activity appears to be involved in Fc receptor mediated stimulation of macrophage function.     

Cloning and mapping of Saccharomyces cerevisiae photoreactivation gene PHR1.

The yeast Saccharomyces cerevisiae, like most organisms, is able to directly repair pyrimidine dimers by using a photoreactivating enzyme and visible light. Cells carrying the phr1 mutation were shown previously to be unable to photoreactivate dimers, but neither the map position nor the primary gene product of the PHR1 gene has been determined. We have cloned this gene and determined its map position. A plasmid containing a 6.4-kilobase yeast DNA insert has been isolated and shown to restore photoreactivation in a phr1 strain. A 3.1-kilobase subclone has also been shown to complement phr1. The original plasmid was targeted to integrate into chromosomal DNA at a site homologous to the insert by cutting within the insert. Two of these integrants have been mapped on the right arm of chromosome XV; the integrants have been further mapped at ca. 13 centimorgans from prt1. It has also been independently determined that phr1 maps at this location. Thus, we have determined the map position of PHR1 and also have shown that the plasmid contains PHR1 rather than a suppressor of the phr1 mutation.