Three-dimensional microscopy of the Rad51 recombination protein during meiotic prophase.

An open question in meiosis is whether the Rad51 recombination protein functions solely in meiotic recombination or whether it is also involved in the chromosome homology search. To address this question, we have performed three-dimensional high-resolution immunofluorescence microscopy to visualize native Rad51 structures in maize male meiocytes. Maize has two closely related RAD51 genes that are expressed at low levels in differentiated tissues and at higher levels in mitotic and meiotic tissues. Cells and nuclei were specially fixed and embedded in polyacrylamide to maintain both native chromosome structure and the three dimensionality of the specimens. Analysis of Rad51 in maize meiocytes revealed that when chromosomes condense during leptotene, Rad51 is diffuse within the nucleus. Rad51 foci form on the chromosomes at the beginning of zygotene and rise to approximately 500 per nucleus by mid-zygotene when chromosomes are pairing and synapsing. During chromosome pairing, we consistently found two contiguous Rad51 foci on paired chromosomes. These paired foci may identify the sites where DNA sequence homology is being compared. During pachytene, the number of Rad51 foci drops to seven to 22 per nucleus. This higher number corresponds approximately to the number of chiasmata in maize meiosis. These observations are consistent with a role for Rad51 in the homology search phase of chromosome pairing in addition to its known role in meiotic recombination.

Evidence for a stromal GTP requirement for the integration of a chlorophyll a/b-binding polypeptide into thylakoid membranes.

The integration of chlorophyll a/b-binding (LHCP) polypeptides and the translocation of the 33-kD oxygen-evolving enhancer protein (OEE33) have been previously shown to occur in chloroplast extracts containing stroma, thylakoids, ATP, and MgCl2. We have re-examined the nucleotide requirement for these two reactions using stromal extract and translation products depleted of low molecular weight compounds. LHCP integration activity was up to 10-fold higher when assayed with GTP compared with ATP, CTP, or UTP. A combination of ATP and GTP supported less LHCP integration activity than GTP alone, suggesting that GTP meets the entire nucleotide requirement. Nonhydrolyzable analogs of GTP were inhibitory, consistent with the idea that GTP hydrolysis is required for integration activity. Periodate-oxidized GTP (GTPox) also inhibited the integration reaction when present during the assay. Pretreatment of stroma with GTPox followed by GTPox removal inhibited integration activity, whereas pretreatment of thylakoids had no effect. We interpret this to mean that a GTP-binding protein involved in integration is localized in the stroma. Translocation of OEE33 was more efficient with ATP than with GTP, and the combination of both nucleotides was not additive. Our data implicate the involvement of a GTPase in LHCP integration but not in the translocation of OEE33.

Characterization of a gene encoding a Ca(2+)-ATPase-like protein in the plastid envelope.

By screening an Arabidopsis expression library with an antiserum against chloroplast envelope proteins, we have isolated a partial cDNA with an open reading frame that encodes a polypeptide similar to P-type cation-transporting ATPases. The corresponding genomic clone was isolated and the complete coding sequence was deduced after identification and mapping of introns. The gene has been designated PEA1 (plastid envelope ATPase) and the predicted polypeptide PEA1p. PEA1p has 946 amino acids and a molecular mass of 104 kDa. This protein is 40-44% identical to various mammalian plasma membrane Ca(2+)-ATPases but lacks the C-terminal calmodulin binding domain present in the mammalian polypeptides. When aligned with mammalian plasma membrane Ca(2+)-ATPases, PEA1p has a 70- to 80-amino acid N-terminal region that extends beyond the N terminus of these enzymes. This extension has some similarity to the transit peptide of the plastid envelope phosphate translocator and may function to target the protein to the plastid. Antibodies raised against a portion of PEA1p recognize a single 90- to 95-kDa polypeptide in chloroplast inner envelope preparations. Transcript abundance as determined by RNase protection was found to be 7- to 9-fold higher in roots than in leaves. Possible roles for a plastid envelope calcium pump are suggested.

Characterization of a chloroplast homologue of the 54-kDa subunit of the signal recognition particle.

Proteins and RNAs homologous to components of the eukaryotic signal recognition particle (SRP) have been identified in a number of prokaryotic organisms. Here we report the isolation of an Arabidopsis thaliana cDNA, FFC (fifty-four chloroplast homologue), that encodes a chloroplast protein (54CP) homologous to the 54-kDa subunit of the signal recognition particle. 54CP shares 27% identity with mammalian SRP54 and 44% identity with the Escherichia coli ffh gene product suggesting a prokaryotic origin for this gene. FFC is a nuclear gene encoding a 62-kDa cytoplasmically synthesized precursor that is capable of being imported into isolated chloroplasts and processed to a 53-kDa stromal polypeptide. Antibodies generated against a portion of 54CP recognize an endogenous 53-kDa chloroplast protein that sediments at 4 S and 70 S, the latter form may be associated with ribosomes. The FFC transcript is most abundant in green shoot tissue whereas etiolated buds and roots have transcript levels about 30 and 10%, respectively, relative to light grown green shoots. By analogy with the eukaryotic signal recognition particle which targets secretory proteins to the endoplasmic reticulum, it is speculated that 54CP may target chloroplast proteins to either the thylakoid or envelope membranes.

Primary structure and characterization of an Arabidopsis thaliana calnexin-like protein.

A cDNA clone (pTE-83) encoding a protein (CNX1p) related to the microsomal Ca(2+)-binding protein, calnexin, was isolated from an Arabidopsis thaliana expression library. Southern and Northern hybridization indicated that CNX1 is a single-copy gene encoding a message of 1900 nucleotides. The open reading frame encodes a polypeptide with 530 amino acids, a molecular mass of 60.5 kDa, and overall 48% identity to dog calnexin. Both animal calnexin and CNX1p contain a large luminal domain followed by a single potential membrane-spanning domain near the C terminus and a small C-terminal domain exposed to the cytoplasm. The in vitro translation product from the cloned cDNA yielded a polypeptide of 67 kDa that was co-translationally imported into dog microsomes and processed to a 64-kDa product. Antibodies generated against the C-terminal half of the protein cross-react with an identically sized protein present in the microsomal fraction from Arabidopsis. Both the imported and native proteins are cleaved by trypsin to a 59-kDa product indicating that the gene product was indeed correctly processed and translocated into dog microsomes and that the membrane topology of CNX1p resembles that of dog calnexin. The presence of a calnexin-like protein within the plant kingdom indicates that this protein is widespread and involved in processes fundamental to all eukaryotes.

Viral plasmacytosis (Aleutian disease) of mink resembling human collagen disease.

A disease in mink has been discovered that has many of the features of collagen diseases in man. Affected animals suffer from wasting with leukopenia and thrombocytopenia as well as plasma cell infiltration, hypergammaglobulinemia, glomerulonephritis, arteritis and amyloidosis. Cell-free filtrates and ultracentrifugates from diseased animals induced the disease in normal mink, and aleutian genotypes were unusually susceptible to infection. This genotype was characterized by abnormal lysosomal structures in all the granule-forming cells, resembling the Chediak-Higashi syndrome of man. Anti-gamma-globulin factors similar to human rheumatoid factors were reported, although tests for antibodies such as ANF and LE factors have been negative. Arteritis and glomerulonephritis lesions stained positively for gamma-globulin, and Coombs-type sensitized red cells have been detected in the majority of affected mink. Some mink develop a monodispersion of hypergammaglobulinemia resembling the serum protein changes in human myeloma. These studies highlight genetic, immunological and microbiological causative factors in a mink disorder resembling human collagen disease.