Mitochondrial DNA insertions into the nuclear chromosomes of the maize Mo17 inbred line [abstract]

Abstract only availableMitochondria contain their own DNA separate from the nucleus; however, most of their genes have been transferred to the nucleus over evolutionary time. The lateral transfer of DNA from the mitochondria to the nucleus appears to be a continuing process and is more frequent in plants than in animals. Our laboratory has documented extensive variation in the nuclear-mitochondrial sequences (NUMTs) among maize inbred lines using total mitochondrial DNA (mtDNA) as probes onto mitotic metaphase chromosomes, a technique known as fluorescence in situ hybridization (FISH). The mitochondrial genome has been cloned into 20 cosmids, which were used to examine the insertions of individual segments. The focus of the current study was to use FISH with the 20 individual mtDNA-containing cosmids to locate mtDNA within the nuclear chromosomes of the Mo17 inbred line of maize and to compare these locations with those of the B73 line. We studied Mo17 because this line and its derivatives are used in crosses with B73-derived lines to create the most commonly used corn hybrids. Fifteen NUMTs had been detected on the Mo17 chromosomes using a mixture of 19 mtDNA-containing cosmids. However, only nine of the 15 NUMTs were seen when applying individual mtDNA-containing cosmids, suggesting that the portions of nuclear DNA corresponding to the individual mtDNA-containing cosmids were too small to be detected until many cosmids were combined. A large NUMT was previously detected on the long arm of chromosome 9 by 14 of the 20 individually tested cosmids in the B73 inbred line. However, a NUMT present at the same site in Mo17 was detected by only 2 of the 20 cosmids (5 and 20). This suggests that the major insertion in B73 is recent and that it may have inserted by homologous recombination

Using a candidate gene approach to identify the restorer-of-fertility Rf4 gene for maize CMS-C

Abstract only availableCytoplasmic male sterility (CMS) is a maternally inherited trait that prevents normal pollen development. Although CMS is a mitochondrial mutation, there are nuclear genes known as restorer-of-fertility genes (Rf) that restore normal pollen development. This makes CMS particularly useful in performing controlled crosses for plant breeding programs and seed production, where the desired female carries a CMS mutation and the male contains the nuclear Rf gene. In maize, CMS type C (CMS-C) can be restored to fertility by the Rf4 gene. Identification of Rf4 can provide insight into the cause of CMS-C pollen abortion, and expand our knowledge of nuclear-mitochondrial communication. Our approach for identifying Rf4 combines genetic information with the physical maps and emerging DNA sequence data. Previous studies have genetically mapped the Rf4 gene to the short arm of a chromosome 8 (Sisco, 1991). Our goal is to identify a candidate gene for Rf4 in the correct region of chromosome 8. Genes in this region were categorized based on the probability of the proteins to be targeted to the mitochondria using MitoPROT and Predotar. The genes were also analyzed for PPR motifs using TPRpred. We used the criteria of mitochondrial targeting and a probable PPR functional motif because most Rf genes identified to date are targeted to mitochondria and have a PPR motif in their sequence. Primers have been designed for candidate genes, and they will be amplified and sequenced from six different maize inbred lines: three with the fertility restoring allele Rf4 and the non-restoring allele rf4. The sequenced genes will be then analyzed for nucleotide polymorphisms that correlate with the Rf4 and rf4 alleles.NSF Plant Genomics Internship @ M

Altered nuclear gene expression in response to mitochondrial mutations in maize [abstract]

Abstract only availableMaize non-chromosomal stripe (NCS) plants are defective plants with mutations within mitochondrial DNA (mtDNA) resulting in deficiencies in the electron transfer chain (ETC). It has been shown the cells will synthesize nuclear proteins in response to the mitochondrial mutations. All NCS mutants are known to up-regulate several stress proteins in response to defective mitochondria, including alternative oxidases and heat shock proteins. Changes in RNA levels can indicate which proteins differ in expression. The NCS2 has a mutation within complex I of the ETC. Using microarrays, total RNA samples of the NCS2 mutant are being compared to those of relatives with normal mitochondria. So far, many differences in RNA levels have been observed. Data is still being collected and being analyzed to determine how many of the apparent differences are statistically significant.MU Monsanto Undergraduate Research Fellowshi

Comparison of regulatory regions in the mitochondrial genomes of grasses

Abstract only availableRegulation of transcription in plant mitochondria is not well understood. The recent sequencing of the mitochondrial genomes of 10 closely related grasses allowed a comparative analysis of regulatory regions. To look for conserved regions and potential “swapped” regulatory regions, we have performed a comparative analysis of the upstream and downstream regions of all of the protein-coding genes in the mitochondrial genomes of eleven grasses: five mitochondrial types of maize (two fertile and three cytoplasmic male sterile), three other taxa within the genus (Zea mays ssp. parviglumis, Z. luxurians, Z. perennis), two close relatives (Tripsacum dactyloides, Sorghum bicolor), and an outgroup, rice. These genomes contain an average of 35 protein-coding genes, composed of 40 transcriptional units. Our analyses examined 1000 base pairs (bp) upstream of the first exon of each transcriptional unit and 1000 bp downstream of its last exon. The reference genome was NB, the most common fertile maize mitochondrial genotype. Compared with the genes from NB, more than half of the mitochondrial genes in the other genomes contain sequences that flank different genes in NB; we refer to these as “swapped” regions. More than 25% of the translocated sequences are longer than 100 bp, and 21 are greater than 500 bp. The longer sequences are more likely to have a regulatory function. In addition, some of these regions were found multiple times: 12 of the translocated gene-flanking regions were found flanking five or more other genes; four had sequences that were flanking ten or more. Furthermore, in Z. luxurians, Z. perennis and T. dactyloides, the co-transcribed 18S and 5S ribosomal RNA genes have been translocated immediately upstream of the start of cox1, with the 5S rRNA 3' end only 80 bp from the start of cox1 exon 1. This is a position that is difficult to rectify with the divergent transcriptional needs of the two types of genes.Plant Genomics Internship @ M

Circular and linear mitochondrial genomes in cytoplasmic male sterile maize [abstract]

Abstract only availableCytoplasmic male sterility (CMS) is a maternally inherited condition in which a plant has an inability to produce viable pollen. It is usually due to the production of a toxic chimeric protein within the mitochondria during the maturation of pollen grains. In maize (Zea mays), there are three types of CMS: CMS-T, CMS-C and CMS-S. The S-type of cytoplasmic male sterility (CMS-S) in maize is associated with the expression of a rearranged mitochondrial DNA region. This CMS-S-specific region includes two co-transcribed chimeric open reading frames, orf355 and orf77. The nuclear restorer-of-fertility gene, Rf3, cleaves all transcripts containing both orfs, including the CMS-S-specific linear 1.6 kb mRNA; this results in male fertility. The Lancaster Surecrop-derived inbred line A619 carries a different and weaker restorer called Rf9. Fertility restoration by Rf3 and Rf9 was compared for their effects upon the CMS-associated region of mitochondrial DNA. Unlike Rf3, Rf9 affects the organization of the CMS-S-specific region. It appears to do this by affecting recombination between linear "S" plasmids and the CMS-S-specific region of the main mitochondrial genome, which produces a linear end from which transcripts for the 1.6 kb mRNA are initiated. By reducing the amount of recombination, Rf9 reduces the amount of linear template available for transcribing the S-associated 1.6 kb RNA. A reduction in this transcript is associated with an increase in pollen survival. We have studied the effects of the two restorer-of-fertility genes from several different inbred lines on the amounts of integrated and linearized orf355/orf77 genes within CMS-S mtDNA.MU Monsanto Undergraduate Research Fellowshi

Mitochondrial DNA insertion into nuclear chromosomes of maize

Abstract only availableEvery mitochondrion contains its own DNA separate from the nucleus. Over evolutionary time, most of the mitochondrial genes have moved to the nucleus so that now mitochondria require nuclear DNA to function. This type of transfer is an apparently ongoing process based on our observations that large pieces of the mitochondrial genome have been transferred to the nucleus. The focus of this study was to find the locations of mitochondrial DNA (mtDNA) on nuclear chromosomes in the B73 line of maize and compare these locations to other lines, using fluorescence in situ hybridization (FISH). First, cosmids previously made from a normal mtDNA genotype (NB) were maxi-prepped and then direct labeled with fluorescent tags. Next we prepared slides of B73 root tips and hybridized the labeled cosmids as well as marker probes to the cells. After hybridization, the slides were viewed and chromosome spread pictures were taken showing the location of the cosmids on the chromosomes. This process was then performed on root tip chromosomes from the Mo17, Black Mexican Sweet (BMS), and B37 lines. Twelve cosmids, representing about 71% of the mitochondrial genome, were examined and 8 different nuclear insertion sites were identified. These dispersed locations were predominantly near centromeres or telomeres on chromosomes 2 and 9. These new findings will make understanding the B73 nuclear genome sequence easier because now researchers will know what to expect at these locations and provide new information about the mechanism of mitochondrial genome transfer to the nucleus.NSF-REU Biology & Biochemistr

Analysis of the mtDNA insertion site on chromosome 9L in maize inbreds using fluorescence in situ hybridization

Abstract only availableAlmost all eukaryotic nuclear genomes show evidence of organellar DNA insertions originating from mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA). While the precise mechanisms of incorporation remain unknown, the phenomenon is frequent and ongoing in many species. In Zea mays, mtDNA insertions differ among inbred lines. A very large mtDNA insertion is found near the centromere of the long arm of chromosome 9 in the B73 inbred. This insertion contains the majority of the mitochondrial genome, while a similarly positioned insertion in the Mo17 inbred line is much smaller. We used recombinant inbred lines from the intermated B73 x Mo17 (IBM) population to determine if the insertions are indeed at the same position. We selected lines with recombination in this region of chromosome 9L. Using two mtDNA probes present in the insertions in both B73 and Mo17, we applied a chromosome painting technique called fluorescence in situ hybridization (FISH) to root-tip metaphase chromosomes and looked for the presence of the mtDNA site on chromosome 9L in the selected IBM lines. If the mtDNA insertion sites in B73 and Mo17 are at different locations, then at least one of the recombinant IBM lines should not display a mtDNA insertion at the chromosome 9 location. However, all of the recombinant IBM lines examined displayed the mtDNA insertion site on chromosome 9L. This indicates that the Mo17 and B73 insertions likely occupy the same region on the chromosome. Furthermore, this suggests that the large mtDNA insertion occurred recently in B73 at a pre-existing site present in both B73 and Mo17.NSF-REU Program in Biological Sciences & Biochemistr

Identification of chloroplast DNA insertions in nuclear chromosomes of maize B73 line using the FISH procedure

Abstract only availableIt is known that chloroplast DNA can incorporate itself into the nuclear genome of plants. However, the sites of chloroplast (ct) DNA integration into chromosomes of maize have not yet been analyzed. This project is the first attempt to find the location of the ctDNA on the maize chromosomes. Fluorescent in situ hybridization is a technique that has proved useful in karyotyping and chromosomal mapping in maize. The FISH procedure is being used in this study to discover the location of the ctDNA in the nuclear genome of the inbred line B37. In order to develop ctDNA “probes” for FISH analysis, we have used the polymerase chain reaction (PCR) to produce fragments of ctDNA. Primers were chosen to amplify fragments of 10 kb or larger. The amplified DNAs were purified and labeled with fluorescent dyes and these probes were subsequently hybridized to chromosomes. The probes recognize and bind to the corresponding DNA sequences within the chromosomes. Root tip cells were used to prepare the slides for hybridization. Because the cells are collected during the metaphase stage of division, the chromosomes are compact and more easily visible. Chromosomes that contain ctDNA can be detected using a compound microscope with fluorescent attachments. The location of the ctDNA on the chromosomes is made visible by the fluorescent labeling of the probe. Eight of eleven regions of the chloroplast genome of the B73 line have been specifically amplified and have been observed under the microscope for FISH analysis. This information will contribute to an understanding of the extent and mechanism of transfer of organellar genomes to the nucleus.MU Monsanto Undergraduate Research Fellowshi

Analysis of mitochondrial DNA insertions into a nuclear chromosome of the maize B73 line

Abstract only availableMitochondrial DNA (mtDNA) is known to have integrated into the nuclear DNA of plants and animals. The purpose of this project is to investigate the on-going migration of mtDNA into the nuclear DNA of maize plants. Specific objectives are to discover the amount of DNA incorporated, whether it is the whole mitochondrial genome or sections, and to see if it has replicated after migration. The maize inbred line B73 has a particularly large mt DNA insert on chromosome 9. Using the fluorescent in situ hybridization (FISH) method, the arrangement of inserted mitochondrial DNA was examined. The FISH method uses fluorescently labeled mtDNA as probes for hybridization to chromosomes. Regions of the chromosomes that contain mtDNA can then be detected using a compound microscope with fluorescent attachments. Locations that contain more mtDNA are brighter. Three combinations of probes that cover different parts of the mitochondrial genome were employed. In order to analyze the arrangement of the DNA, the chromosomes were prepared from a stage of meiosis called pachynema in which the chromosomes are elongated and have not yet begun to condense. The results have confirmed the presence of all three probes within the large insertion of mtDNA on chromosome 9 of B73. The data suggest that either different parts of the mitochondrial genome are incorporated preferentially or that there is selective replication of portions of the mitochondrial genome after incorporation.MU Monsanto Undergraduate Research Fellowshi