SummaryAlthough double fertilization in angiosperm was discovered in 1898, we still know nothing about the proteins that mediate gamete recognition and fusion in plants. Because sperm are small and embedded within the large vegetative cell of the pollen grain, mRNAs from sperm are poorly represented in EST databases. We optimized¯uorescence-activated cell sorting (FACS) in order to isolate Zea mays sperm free of contaminating vegetative cell cytoplasm, and constructed a cDNA library. Sequencing of over 1100 cDNAs from the unampli®ed library revealed that sperm have a diverse complement of mRNAs. Most transcripts were singletons; the most abundant was sequenced only 17 times. About 8% of the sequences are predicted to encode secreted or plasma membrane-localized proteins and are therefore candidates that might mediate gamete interactions. About 8% of the sequences correspond to retroposons. Plant sperm have condensed chromatin and are thought to be transcriptionally inactive. We used RT-PCR and in situ hybridization to determine when selected sperm mRNAs were transcribed. Sperm transcripts encoding proteins involved in general cell functions were present throughout pollen development and were more abundant in tricellular pollen than in sperm cells, suggesting that these transcripts were also present in the larger vegetative cell. However, several transcripts, which encode proteins that are most similar to hypothetical Arabidopsis proteins, appeared to be present exclusively in the sperm cells inside mature pollen, but were already present in unicellular microspores. This suggests that certain transcripts might be transcribed early during pollen development and later partitioned into the sperm cells.
Previously, in an effort to better understand the male contribution to fertilization, we completed a maize (Zea mays) sperm expressed sequence tag project. Here, we used this resource to identify promoters that would direct gene expression in sperm cells. We used reverse transcription-polymerase chain reaction to identify probable sperm-specific transcripts in maize and then identified their best sequence matches in the Arabidopsis (Arabidopsis thaliana) genome. We tested five different Arabidopsis promoters for cell specificity, using an enhanced green fluorescent protein reporter gene. In pollen, the AtGEX1 (At5g55490) promoter is active in the sperm cells and not in the progenitor generative cell or in the vegetative cell, but it is also active in ovules, roots, and guard cells. The AtGEX2 (At5g49150) promoter is active only in the sperm cells and in the progenitor generative cell, but not in the vegetative cell or in other tissues. A third promoter, AtVEX1 (At5g62580), was active in the vegetative cell during the later stages of pollen development; the other promoters tested (At1g66770 and At1g73350) did not function in pollen. Comparisons among GEX1 and GEX2 homologs from maize, rice (Oryza sativa), Arabidopsis, and poplar (Populus trichocarpa) revealed a core binding site for Dof transcription factors. The AtGEX1 and AtGEX2 promoters will be useful for manipulating gene expression in sperm cells, for localization and functional analyses of sperm proteins, and for imaging of sperm dynamics as they are transported in the pollen tube to the embryo sac.
Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomatids, consists of thousands of minicircles and 20 to 30 maxicircles catenated into a single large network and exists in the cell as a highly organized compact disc structure. To investigate the role of kinetoplast-associated proteins in organizing and condensing kDNA networks into this disc structure, we have cloned three genes encoding kinetoplast-associated proteins. The KAP2, KAP3, and KAP4 genes encode proteins p18, p17, and p16, respectively. These proteins are small basic proteins rich in lysine and alanine residues and contain 9-amino-acid cleavable presequences. Proteins p17 and p18 are closely related to each other, with 48% identical residues and carboxyl tails containing almost exclusively lysine, alanine, and serine or threonine residues. These proteins have been expressed as Met-His 6 -tagged recombinant proteins and purified by metal chelate chromatography. Each of the recombinant proteins is capable of compacting kDNA networks in vitro and was shown to bind preferentially to a specific fragment of minicircle DNA. Expression of each of these proteins in an Escherichia coli mutant lacking the HU protein rescued a defect in chromosome condensation and segregation in the mutant cells and restored a near-normal morphological appearance. Proteins p16, p17, and p18 have been localized within the cell by immunofluorescence methods and appear to be present throughout the kDNA. Electron-microscopic immunolocalization of p16 shows that p16 is present both within the kDNA disc and in the mitochondrial matrix at opposite edges of the kDNA disc. Our results suggest that nucleus-encoded H1-like proteins may be involved in the organization and segregation of kDNA networks in trypanosomatids.The mitochondrial DNA of kinetoplastid protozoa consists of about 5,000 minicircles and 20 to 30 maxicircles. These circular DNAs are held together by catenation into a single two-dimensional sheet of DNA referred to as a kinetoplast DNA (kDNA) network. The minicircles exist within the network as relaxed covalently closed circles, with each minicircle linked on average to three other minicircles (3). Each cell has only one such network, which in purified form or in cell lysates has a diameter similar in size (8 to 10 m) to that of the whole cell. In vivo, the kDNA network exists as a highly condensed disc about 1 m in diameter and approximately 0.4 m thick (25; also see below). The kDNA disc is physically associated with the basal body of the cell and is oriented with the axis of the disc parallel to that of the flagellum. Electron micrographs of sections through the kDNA disc also show DNA fibers oriented parallel to the axis of the disc. For recent reviews of the structure and replication of kinetoplast DNA, see references 7 and 24.We have developed methods recently for identifying and characterizing proteins that may play a role in organizing and condensing the kDNA network into the compact disc structure observed in vivo (35). Proteins bound to kDNA are covalently...
The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata has an unusual structure composed of minicircles and maxicircles topologically interlocked into a single network and organized in a disc-shaped structure at the base of the f lagellum. We previously purified a structure-specific endonuclease (SSE1), based on its RNase H activity, that is enriched in isolated kinetoplasts. The endonuclease gene has now been cloned, sequenced, and found to be closely related to the 5 exonuclease domain of bacterial DNA polymerase I proteins. Although the protein does not contain a typical mitochondrial leader sequence, the enzyme is shown to colocalize with a type II DNA topoisomerase and a DNA polymerase  at antipodal sites f lanking the kinetoplast disc. Cell synchronization studies with an epitope-tagged construct show that the localization of the endonuclease to the antipodal sites varies in a cell The trypanosomatid Crithidia fasciculata has an unusual mitochondrial DNA structure called the kinetoplast DNA, which is composed of 5,000 minicircles of 2.5 kilobases (kb) and 25 maxicircles of 37 kb catenated into a single network. The maxicircles encode conventional mitochondrial proteins and ribosomal RNAs. Unlike most mitochondrial DNA, kinetoplast DNA replicates only once in the cell cycle in approximate synchrony with nuclear DNA replication (1, 2). Minicircles replicate free of the network as intermediates (3). Lightstrand synthesis is RNA primed at each of the two universal minicircle sequences and occurs continuously (4-6). Heavystrand synthesis occurs discontinuously via Okazaki-like fragments that are also likely to be RNA primed (5, 7). Nicks and gaps remaining in newly replicated minicircles are partially repaired before reattachment to the network but are not completely repaired until just before network division into two daughter networks. (4,5,8,9).Several enzymes involved in kinetoplast DNA replication have been purified and localized within the kinetoplast. A primase has been localized to a region adjacent to the two faces of the kinetoplast disk, suggesting that RNA priming and the early stages of minicircle replication may occur in this region (10). Additionally, a topoisomerase II and a DNA polymerase  have been localized to two antipodal sites flanking the kinetoplast disc where the minicircles are reattached to the network (11-14). Minicircle replication intermediates have also been detected at these sites (11). The low processivity of polymerase  suggests that it may be involved in the repair of gaps remaining in newly replicated minicircles. The topoisomerase II is likely to mediate minicircle reattachment after partial gap repair.To further define the components of the replication machinery and their localization relative to the kinetoplast disc, we have sought to identify and purify additional kinetoplast replication proteins. The purification and characterization of a structure-specific endonuclease (SSE1) enriched in the kinetoplast fraction of C. fasciculata ...
The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata consists of minicircles and maxicircles topologically interlocked in a single network per cell. Individual minicircles replicate unidirectionally from either of two replication origins located 180 degrees apart on the minicircle DNA. Initiation of minicircle leading-strand synthesis involves the synthesis of an RNA primer which is removed in the last stage of replication. We report here the purification to near homogeneity of a structure-specific DNA endo-nuclease based on the RNase H activity of the enzyme on a poly(rA).poly(dT) substrate. RNase H activity gel analysis of whole cell and kinetoplast extracts shows that the enzyme is enriched in kinetoplast fractions. The DNA endonuclease activity of the enzyme is specific for DNA primers annealed to a template strand and requires an unannealed 5' tail. The enzyme cleaves 3' of the first base paired nucleotide releasing the intact tail. The purified enzyme migrates as a 32 kDa protein on SDS gels and has a Stoke's radius of 21.5 A and a sedimentation coefficient of 3.7 s, indicating that the protein is a monomer in solution with a native molecular mass of 32.4 kDa. These results suggest that the enzyme may be involved in RNA primer removal during minicircle replication.
The Crithidia fasciculata RNH1 gene encodes an RNase H, an enzyme that specifically degrades the RNA strand of RNA-DNA hybrids. The RNH1 gene is contained within an open reading frame (ORF) predicted to encode a protein of 53.7 kDa. Previous work has shown that RNH1 expresses two proteins: a 38 kDa protein and a 45 kDa protein which is enriched in kinetoplast extracts. Epitope tagging of the C-terminus of the RNH1 gene results in localization of the protein to both the kinetoplast and the nucleus. Translation of the ORF beginning at the second in-frame methionine codon predicts a protein of 38 kDa. Insertion of two tandem stop codons between the first ATG codon and the second in-frame ATG codon of the ORF results in expression of only the 38 kDa protein and the protein localizes specifically to the nucleus. Mutation of the second methionine codon to a valine codon prevents expression of the 38 kDa protein and results in exclusive production of the 45 kDa protein and localization of the protein only in the kinetoplast. These results suggest that the kinetoplast enzyme results from processing of the full-length 53.7 kDa protein. The nuclear enzyme appears to result from translation initiation at the second in-frame ATG codon. This is the first example in trypanosomatids of the production of nuclear and mitochondrial isoforms of a protein from a single gene and is the only eukaryotic gene in the RNase HI gene family shown to encode a mitochondrial RNase H.
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