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J L Bennetzen

Publications and source records attributed to J L Bennetzen.

At least 19 recordsLinked to original sources

Grass genomes.

For the most part, studies of grass genome structure have been limited to the generation of whole-genome genetic maps or the fine structure and sequence analysis of single genes or gene clusters. We have investigated large contiguous segments of the genomes of maize, sorghum, and rice, primarily focusing on intergenic spaces. Our data indicate that much (>50%) of the maize genome is composed of interspersed repetitive DNAs, primarily nested retrotransposons that insert between genes. These retroelements are less abundant in smaller genome plants, including rice and sorghum. Although 5- to 200-kb blocks of methylated, presumably heterochromatic, retrotransposons flank most maize genes, rice and sorghum genes are often adjacent. Similar genes are commonly found in the same relative chromosomal locations and orientations in each of these three species, although there are numerous exceptions to this collinearity (i.e., rearrangements) that can be detected at the levels of both the recombinational map and cloned DNA. Evolutionarily conserved sequences are largely confined to genes and their regulatory elements. Our results indicate that a knowledge of grass genome structure will be a useful tool for gene discovery and isolation, but the general rules and biological significance of grass genome organization remain to be determined. Moreover, the nature and frequency of exceptions to the general patterns of grass genome structure and collinearity are still largely unknown and will require extensive further investigation.

Journal Article

Matrix attachment regions and structural colinearity in the genomes of two grass species.

In order to gain insights into the relationship between spatial organization of the genome and genome function we have initiated studies of the co-linear Sh2/A1- homologous regions of rice (30 kb) and sorghum (50 kb). We have identified the locations of matrix attachment regions (MARs) in these homologous chromosome segments, which could serve as anchors for individual structural units or loops. Despite the fact that the nucleotide sequences serving as MARs were not detectably conserved, the general organizational patterns of MARs relative to the neighboring genes were preserved. All identified genes were placed in individual loops that were of comparable size for homologous genes. Hence, gene composition, gene orientation, gene order and the placement of genes into structural units has been evolutionarily conserved in this region. Our analysis demonstrated that the occurrence of various 'MAR motifs' is not indicative of MAR location. However, most of the MARs discovered in the two genomic regions were found to co-localize with miniature inverted repeat transposable elements (MITEs), suggesting that MITEs preferentially insert near MARs and/or that they can serve as MARs.

Base Composition

Sequence organization and conservation in sh2/a1-homologous regions of sorghum and rice.

Previously, we have demonstrated microcolinearity of gene composition and orientation in sh2/a1-homologous regions of the rice, sorghum, and maize genomes. However, the sh2 and a1 homologues are only about 20 kb apart in both rice and sorghum, while they are separated by about 140 kb in maize. In order to further define sequence organization and conservation in sh2/a1-homologous regions, we have completely sequenced a 42,446-bp segment of sorghum DNA. Four genes were identified: a homologue of sh2, two homologues of a1, and a putative transcriptional regulatory gene. A solo long terminal repeat of the retroelement Leriathan was detected between the two a1 homologues, and eight miniature inverted repeat transposable elements were found in this region. Comparison of the sorghum sequence with the sequence of the homologous segment from rice indicated that only the identified genes were evolutionarily conserved between these two species, which have evolved independently for over 50 million years. The introns of the a1 homologues have evolved faster than the introns of the sh2 homologue. The a1 tandem duplication appears to be an ancient event that may have preceded the ancestral divergence of maize, sorghum, and rice.

Amino Acid Sequence

Microcolinearity in sh2-homologous regions of the maize, rice, and sorghum genomes.

Large regions of genomic colinearity have been demonstrated among grass species by recombinational mapping, but the degree of chromosomal conservation at the sub-centimorgan level has not been extensively investigated. We cloned the rice and sorghum genes homologous to the sh2 locus of maize on bacterial artificial chromosomes (BACs), and observed that a homologue of the maize a1 gene was also present on each of these BACs. In sorghum, we found a direct duplication of a1 homologues separated by about 10 kb. In maize, sh2 and a1 are approximately 140 kb apart and transcribed in the same direction, with sh2 upstream of a1. In rice and sorghum, this arrangement is fully conserved. However, the sh2 and a1 homologues are separated by about 19 kb in both rice and sorghum. We found low-copy-number and repetitive DNAs between the sh2 and a1 homologues of sorghum and rice. The sh2 and a1 homologues cross-hybridized, but the repetitive DNA and most low-copy-number sequences between these genes did not. These results indicate that maize, sorghum, and rice have conserved gene order and composition in the sh2-a1 region, but have acquired extensive qualitative and quantitative differences in the sequences between these genes.

Base Sequence

Identification of a pathogenicity locus, rpfA, in Erwinia carotovora subsp. carotovora subsp. carotovora that encodes a two-component sensor-regulator protein.

A mutant of Erwinia carotovora subsp. carotovora, AH2552, created by a Mud1 insertion was found to be reduced in plant pathogenicity and deficient in extracellular protease and cellulase activity, although it produced normal levels of pectate lyase and polygalacturonase. A cosmid clone, pEC462, was isolated from a wild-type E. carotovora subsp. carotovora DNA library that concomitantly restored pathogenicity and protease and cellulase activities of AH2552 to wild-type levels when present in trans. The genetic locus that was disrupted in AH2552 by insertion of Mud1 has been designated rpfA, for regulator of pathogenicity factors. Sequencing of the rpfA region identified an open reading frame of 2,787 bp, and the predicted 929-amino acid polypeptide shared high identity with several two-component sensor-regulator proteins: BarA from Escherichia coli, ApdA from Pseudomonas fluorescens, PheN from P. tolaasii, RepA from P. viridiflava, LemA from P. syringae pv. syringae, and RpfC from Xanthomonas campestris pv. campestris. The RpfA locus described in this study encodes a putative sensor kinase protein that is involved in both extracellular protease and cellulase production and the pathogenicity of E. carotovora subsp. carotovora on potato tubers.

Aconitate Hydratase

Nested retrotransposons in the intergenic regions of the maize genome.

The relative organization of genes and repetitive DNAs in complex eukaryotic genomes is not well understood. Diagnostic sequencing indicated that a 280-kilobase region containing the maize Adh1-F and u22 genes is composed primarily of retrotransposons inserted within each other. Ten retroelement families were discovered, with reiteration frequencies ranging from 10 to 30,000 copies per haploid genome. These retrotransposons accounted for more than 60 percent of the Adh1-F region and at least 50 percent of the nuclear DNA of maize. These elements were largely intact and are dispersed throughout the gene-containing regions of the maize genome.

Chromosomes, Artificial, Yeast

Gene identification in a complex chromosomal continuum by local genomic cross-referencing.

Most higher plants have complex genomes containing large quantities of repetitive DNA interspersed with low-copy-number sequences. Many of these repetitive DNAs are mobile and have homology to RNAs in various cell types. This can make it difficult to identify the genes in a long chromosomal continuum. It was decided to use genic sequence conservation and grass genome co-linearity as tools for gene identification. A bacterial artificial chromosome (BAC) clone containing sorghum genomic DNA was selected using a maize Adh1 probe. The 165 kb sorghum BAC was tested for hybridization to a set of clones representing the contiguous 280 kb of DNA flanking maize Adh1. None of the repetitive maize DNAs hybridized, but most of the low-copy-number sequences did. A low-copy-number sequence that did cross-hybridize was found to be a gene, while one that did not was found to be a low-copy-number retrotransposon that was named Reina. Regions of cross-hybridization were co-linear between the two genomes, but closer together in the smaller sorghum genome. These results indicate that local genomic cross-referencing by hybridization of orthologous clones can be an efficient and rapid technique for gene identification and studies of genome organization.

Alcohol Dehydrogenase

The y1 gene of maize codes for phytoene synthase.

The cloned y1 locus of maize was sequenced and found to encode phytoene synthase. Different "wild-type" alleles of the locus were found to differ by the insertion of transposable elements in their promoter and polyA addition regions, and by the length of a CCA tandem repeat series, without any obvious effect on function of the gene. A dominant Y1 ("wild-type") allele was observed to be expressed at highest levels in the seedling but also in the embryo and endosperm. The Mu3 transposable element insertion responsible for a pastel allele of y1, which gives lowered levels of carotenoids in the endosperm of kernels and seedlings grown at high temperatures, was located in the 5' end of the gene. Although the size of the transcript from this y1 mutation suggests that the Mu3 element provides the promoter for this allele, leaf tissue in this mutant line contained approximately normal amounts of y1 mRNA. A recessive allele of y1, which conditions normal levels of carotenoids in the embryo and seedling, but almost no carotenoids in the endosperm, was found to accumulate normal amounts of y1 mRNA in the seedling and embryo, while y1 transcripts were not detected in the endosperm.

Alkyl and Aryl Transferases

Regional and racial specificities in sorghum germplasm assessed with DNA markers.

Three different molecular marker technologies were used to determine the relatedness of 84 different lines of sorghum. Both racial characterization and geographical origin were found to be correlated with relatedness. In some cases, the region of origin was the more significant factor, where samples of different races from the same locality were more closely related than were samples of the same race from different localities. Wild sorghums were shown to have few novel alleles, suggesting that they would be poor sources of germplasm diversity. The results also indicated that Chinese sorghums are a narrow and distinctive group that is most closely related to race bicolor.

Base Sequence

The Hybaid Lecture. Microcollinearity and segmental duplication in the evolution of grass nuclear genomes.

Recent studies have shown that grass genomes have very similar gene compositions and regions of conserved gene order, as exemplified by collinear genetic maps of DNA markers. We have begun the detailed study of sequence organization in large (100-500 kb) segments of the nuclear genomes of maize, sorghum and rice. Our results indicate collinearity of genes in the regions homoeologous to the maize adh1 and sh2-a1 genes. Comparable genes were found to be physically closer to each other in grasses with small genomes (rice and sorghum) than they are in maize. In several instances, we have found evidence of tandem and 'distantly tandem' duplications of segments containing maize and sorghum genes. These duplications complicate characterizations of microcollinearity and could also interfere with some map-based approaches to gene isolation.

Biological Evolution

Mu1-related transposable elements of maize preferentially insert into low copy number DNA.

The Mutator transposable element system of maize was originally identified through its induction of mutations at an exceptionally high frequency and at a wide variety of loci. The Mu1 subfamily of transposable elements within this system are responsible for the majority of Mutator-induced mutations. Mu 1-related elements were isolated from active Mutator plants and their flanking DNA was characterized. Sequence analyses revealed perfect nine base target duplications directly flanking the insert for 13 of the 14 elements studied. Hybridizational studies indicated that Mu1-like elements insert primarily into regions of the maize genome that are of low copy number. This preferential selection of low copy number DNA as targets for Mu element insertion was not directed by any specific secondary structure(s) that could be detected in this study, but the 9-bp target duplications exhibited a discernibly higher than random match with the consensus sequence 5'-G-T-T-G-G/C-A-G-G/A-G-3'.

Chromosome Mapping

New rust resistance specificities associated with recombination in the Rp1 complex in maize.

We address the question of whether genetic reassortment events, including unequal crossing over and gene conversion, at the Rp1 complex are capable of generating novel resistance specificities that were not present in the parents. Some 176 events involving genetic reassortment within the Rp1 complex were screened for novel resistance specificities with a set of 11 different rust biotypes. Most (150/176) of the events were susceptible to all tested rust biotypes, providing no evidence for new specificities. Eleven events selected as double-resistant recombinants, when screened with the 11 test biotypes, showed the combined resistance of the two parental types consistent with a simple recombination and pyramiding of the parental resistances. Nine events selected either as having partial resistance or complete susceptibility to a single biotype possessed resistance to a subset of the biotypes that the parents were resistant to, suggesting segregation of resistance genes present in the parental Rp1 complex. Four events gave rise to novel specificities being resistant to at least one rust biotype to which both parents were susceptible. All four had flanking marker exchange, demonstrating that crossing over within the Rp1 complex is associated with the appearance of new rust resistance specificities.

Basidiomycota

Matrix attachment regions and transcribed sequences within a long chromosomal continuum containing maize Adh1.

We provide evidence for the location of matrix attachment sites along a contiguous region of 280 kb on maize chromosome 1. We define nine potential loops that vary in length from 6 kb to > 75 kb. The distribution of the different classes of DNA within this continuum with respect to the predicted structural loops reveals an interesting correlation: the long stretches of mixed classes of highly repetitive DNAs are often segregated into topologically sequestered units, whereas low-copy-number DNAs (including the alcohol dehydrogenase1 [adh1] gene) are positioned in separate loops. Contrary to expectations, several classes of highly repeated elements with representatives in this region were found to be transcribed, and some of these exhibited tissue-specific patterns of expression.

Alcohol Dehydrogenase

Assessment of genome origins and genetic diversity in the genus Eleusine with DNA markers.

Finger millet (Eleusine coracana), an allotetraploid cereal, is widely cultivated in the arid and semiarid regions of the world. Three DNA marker techniques, restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), and inter simple sequence repeat amplification (ISSR), were employed to analyze 22 accessions belonging to 5 species of Eleusine. An 8 probe--3 enzyme RFLP combination, 18 RAPD primers, and 6 ISSR primers, respectively, revealed 14, 10, and 26% polymorphism in 17 accessions of E. coracana from Africa and Asia. These results indicated a very low level of DNA sequence variability in the finger millets but did allow each line to be distinguished. The different Eleusine species could be easily identified by DNA marker technology and the 16% intraspecific polymorphism exhibited by the two analyzed accessions of E. floccifolia suggested a much higher level of diversity in this species than in E. coracana. Between species, E. coracana and E. indica shared the most markers, while E. indica and E. tristachya shared a considerable number of markers, indicating that these three species form a close genetic assemblage within the Eleusine. Eleusine floccifolia and E. compressa were found to be the most divergent among the species examined. Comparison of RFLP, RAPD, and ISSR technologies, in terms of the quantity and quality of data output, indicated that ISSRs are particularly promising for the analysis of plant genome diversity.

Base Sequence