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.

Retrotransposons, transposable elements related to animal retroviruses, are found in all eukaryotes investigated and make up the majority of many plant genomes. Their ubiquity points to their importance, especially in their contribution to the large-scale structure of complex genomes. The nature and frequency of retro-element appearance, activation and amplification are poorly understood in all higher eukaryotes. Here we employ a novel approach to determine the insertion dates for 17 of 23 retrotransposons found near the maize adh1 gene, and two others from unlinked sites in the maize genome, by comparison of long terminal repeat (LTR) divergences with the sequence divergence between adh1 in maize and sorghum. All retrotransposons examined have inserted within the last six million years, most in the last three million years. The structure of the adh1 region appears to be standard relative to the other gene-containing regions of the maize genome, thus suggesting that retrotransposon insertions have increased the size of the maize genome from approximately 1200 Mb to 2400 Mb in the last three million years. Furthermore, the results indicate an increased mutation rate in retrotransposons compared with genes.

Orthologous adh regions of the sorghum and maize genomes were sequenced and analyzed. Nine known or candidate genes, including adh1, were found in a 225-kilobase (kb) maize sequence. In a 78-kb space of sorghum, the nine homologues of the maize genes were identified in a colinear order, plus five additional genes. The major fraction of DNA in maize, occupying 166 kb (74%), is represented by 22 long terminal repeat (LTR) retrotransposons. About 6% of the sequence belongs to 33 miniature inverted-repeat transposable elements (MITEs), remnants of DNA transposons, 4 simple sequence repeats, and low-copy-number DNAs of unknown origin. In contrast, no LTR retroelements were detected in the orthologous sorghum region. The unconserved sorghum DNA is composed of 20 putative MITEs, transposon-like elements, 5 simple sequence repeats, and low-copy-number DNAs of unknown origin. No MITEs were discovered in the 166 kb of DNA occupied by the maize LTR retrotransposons. In both species, MITEs were found in the space between genes and inside introns, indicating specific insertion and͞or retention for these elements. Two adjacent sorghum genes, including one gene missing in maize, had colinear homologues on Arabidopsis chromosome IV, suggesting two rearrangements in the sorghum and three in the maize genome in comparison to a four-gene region of Arabidopsis. Hence, multiple small rearrangements may be present even in largely colinear genomic regions. These studies revealed a much higher degree of diversity at a microstructural level than predicted by genetic mapping studies for closely related grass species, as well as for comparisons of monocots and dicots.The grasses belong to a family of monocotyledonous angiosperms that are well differentiated morphologically from the other angiosperm families and have a single (monophyletic) origin. Their genome sizes, however, may vary a great deal between species. Thus, rice has an estimated genome size of 430 megabases, which is Ϸ11ϫ smaller than barley, 6ϫ smaller than maize, and 2ϫ smaller than sorghum. These large differences in genome sizes, coupled with differences in the degree and the nature of their investigations, have obscured some common features of grass genomic design. Recent studies comparing high-density linkage maps with DNA markers revealed extensive synteny of chromosomal segments between related species (1-5). Valuable as it is, full genome recombinational mapping of DNA markers is not an efficient approach for detecting small rearrangements. Because the available high-resolution maps based on completed nucleotide sequence are largely restricted to individual genes and their proximal neighborhoods, we are left with two obvious questions that cannot be answered at a full-genome level of analysis. These questions are, will the colinearity observed at the 2-to 20-centimorgan level, the sensitivity level of standard recombinational mapping, be preserved or will it break down at a local level (5), and what will the pattern of gene distribution be, relative to the no...

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 f lank 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.Very little is known about the structure of the nuclear genomes of higher plants, although comprehensive investigations are now underway into the sequence composition of the unusually small [about 110-megabase pair (mbp)] genome of Arabidopsis thaliana. Most plant nuclei contain more than five times as much DNA as that of Arabidopsis, ranging up to the over 110,000 mbp of Fritillaria assyriaca (1). Part of this genome size variation is caused by differences in ploidy, but the majority is caused by differences in the amounts of repetitive DNA (2). Some of these repetitive sequences are found in tandemly repeated satellites, like the chromosomal knobs of maize (3), but most are represented by interspersed repeats that vary in copy number from tens to hundreds to thousands per haploid nucleus (4). The nature and organization of these repeats, and their functional or structural relationship to genes, are not well understood.Low-density genetic maps, including those for several cereal species (5-7), have shown that conserved DNA markers (primarily genes) often are found in the same linear order in different plant species. This discovery of the collinearity of ''orthologous'' genes (i.e., those with a direct evolutionarily relationship) in cereals has allowed a wholly new perspective on how genes and information can be used synergistically in the study and improvement of all grasses (8-10). Although many exceptions to collinearity exist in these comparative genetic maps (5, 7, 10), collinearity suppli...

Monkeypox is a zoonotic viral disease that occurs primarily in Central and West Africa. A recent outbreak in the United States heightened public health concerns for susceptible human populations. Vaccinating with vaccinia virus to prevent smallpox is also effective for monkeypox due to a high degree of sequence conservation. Yet, the identity of antigens within the monkeypox virus proteome contributing to immune responses has not been described in detail. We compared antibody responses to monkeypox virus infection and human smallpox vaccination by using a protein microarray covering 92–95% (166–192 proteins) of representative proteomes from monkeypox viral clades of Central and West Africa, including 92% coverage (250 proteins) of the vaccinia virus proteome as a reference orthopox vaccine. All viral gene clones were verified by sequencing and purified recombinant proteins were used to construct the microarray. Serum IgG of cynomolgus macaques that recovered from monkeypox recognized at least 23 separate proteins within the orthopox proteome, while only 14 of these proteins were recognized by IgG from vaccinated humans. There were 12 of 14 antigens detected by sera of human vaccinees that were also recognized by IgG from convalescent macaques. The greatest level of IgG binding for macaques occurred with the structural proteins F13L and A33R, and the membrane scaffold protein D13L. Significant IgM responses directed towards A44R, F13L and A33R of monkeypox virus were detected before onset of clinical symptoms in macaques. Thus, antibodies from vaccination recognized a small number of proteins shared with pathogenic virus strains, while recovery from infection also involved humoral responses to antigens uniquely recognized within the monkeypox virus proteome.