Maize individualized chromosome and derived radiation hybrid lines and their use in functional genomics

The duplicated and rearranged nature of plant genomes frequently complicates identification, chromosomal assignment and eventual manipulation of DNA segments. Separating an individual chromosome from its native complement by adding it to an alien genetic background together with the generation of radiation hybrids from such an addition line can enable or simplify structural and functional analyses of complex duplicated genomes. We have established fertile disomic addition lines for each of the individual maize chromosomes, except chromosome 10, with oat as the host species; DNA is available for chromosome 10 in a haploid oat background. We report on instability and transmission in disomic additions of maize chromosomes 1, 5, and 8; the chromosome 2, 3, 4, 6, 7, and 9 additions appear stable. The photoperiodic response of the two recovered maize chromosome 1 addition lines contrasts to the long-day flowering response of the oat parents and the other addition lines. Only when grown under short days did maize chromosome 1 addition lines set seed, and only one line transmitted the maize chromosome 1 to offspring. Low resolution radiation hybrid maps are presented for maize chromosomes 2 and 9 to illustrate the use of radiation hybrids for rapid physical mapping of large numbers of DNA sequences, such as ESTs. The potential of addition and radiation hybrid lines for mapping duplicated sequences or gene families to chromosome segments is presented and also the use of the lines to test interactions between genes located on different maize chromosomes as observed for ectopic expression of cell fate alterations. Electronic Publication     

Phylogenetic tests of the hypothesis of block duplication of homologous genes on human chromosomes 6, 9, and 1

There are 10 gene families that have members on both human chromosome 6 (6p21.3, the location of the human major histocompatibility complex [MHC]) and human chromosome 9 (mostly 9q33-34). Six of these families also have members on mouse chromosome 17 (the mouse MHC chromosome) and mouse chromosome 2. In addition, four of these families have members on human chromosome 1 (1q21-25 and 1p13), and two of these have members on mouse chromosome 1. One hypothesis to explain these patterns is that members of the 10 gene families of human chromosomes 6 and 9 were duplicated simultaneously as a result of polyploidization or duplication of a chromosome segment ("block duplication"). A subsequent block duplication has been proposed to account for the presence of representatives of four of these families on human chromosome 1. Phylogenetic analyses of the 9 gene families for which data were available decisively rejected the hypothesis of block duplication as an overall explanation of these patterns. Three to five of the genes on human chromosomes 6 and 9 probably duplicated simultaneously early in vertebrate history, prior to the divergence of jawed and jawless vertebrates, and shortly after that, all four of the genes on chromosomes 1 and 9 probably duplicated as a block. However, the other genes duplicated at different times scattered over at least 1.6 billion years. Since the occurrence of these clusters of related genes cannot be explained by block duplication, one alternative explanation is that they cluster together because of shared functional characteristics relating to expression patterns.      

The comparison of intrinsic rates of increase among chromosome-substituted lines resistant and susceptible to organophosphate insecticides in Drosophila melanogaster

To investigate the genetic basis of the seasonal fluctuations in resistance to three organophosphates, observed within a natural population of Drosophila melanogaster (Meigen), we compared the intrinsic rate of increase, generation time and net reproduction rate among chromosome substitution lines derived from a resistant and a susceptible line, obtained from this natural population. There was significant variation among substituted lines; lines possessing the third chromosome from the resistant line, which confers resistance to the three organophosphates, generally showed lower mean values of these fitness measures. Chromosomal analyses also indicated significant negative contributions of the third chromosome from the resistant line. However, significant positive contributions of the interactions among chromosomes from the resistant line to these fitness measures were also detected. We further conducted a local stability analysis, in which each chromosome-substituted line was assumed to be introduced at a low frequency into the initial susceptible population. It was demonstrated that the resistance factor(s) on the third chromosome tend to decrease in their frequency under both density-independent and juvenile density-regulated conditions. Based on these results, a possible explanation for the seasonal fluctuations in resistance to the three organophosphates observed in the natural population was proposed.     

Disomic Thinopyrum intermedium addition lines in wheat with barley yellow dwarf virus resistance and with rust resistances.

Zhong 5 is a partial amphiploid (2n = 56) between Triticum aestivum (2n = 42) and Thinopyrum intermedium (2n = 42) carrying all the chromosomes of wheat and seven pairs of chromosomes from Th. intermedium. Following further backcrossing to wheat, six independent stable 2n = 44 lines were obtained representing 4 disomic chromosome addition lines. One chromosome confers barley yellow dwarf virus (BYDV) resistance, whereas two other chromosomes carry leaf and stem rust resistance; one of the latter also confers stripe rust resistance. Using RFLP and isozyme markers we have shown that the extra chromosome in the Zhong 5-derived BYDV resistant disomic addition lines (Z1, Z2, or Z6) belongs to the homoeologous group 2. It therefore carries a different locus to the BYDV resistant group 7 addition, L1, described previously. The leaf, stem, and stripe rust resistant line (Z4) carries an added group 7 chromosome. The line Z3 has neither BYDV nor rust resistance, is not a group 2 or group 7 addition, and is probably a group 1 addition. The line Z5 is leaf and stem rust resistant, is not stripe rust resistant, and its homoeology remains unknown.     

Tandem duplication of the NF1 gene detected by high-resolution FISH in the 17q11.2 region

The gene for neurofibromatosis type 1 (NF1), mapping to 17q11.2, has one of the highest observed mutation rates, partially because of its large size and gene conversion primed by NF1 pseudogenes. We have previously shown by means of high resolution fluorescence in situ hybridization (FISH) that a number of the loci flanking the NF1 gene are duplicated, in agreement with the reported presence of NF1 repetitive sequences (REPs). We report a direct tandem duplication of the NF1 gene identified in 17q11.2 by high-resolution FISH. FISH on stretched chromosomes with locus-specific probes revealed the duplication of the NF1 gene from the promoter to 3'UTR, but with at least the absence of exon 22. Fiber FISH with P1 artificial and bacterial artifical chromosomes, including the NF1 5'UTR and 3'UTR and flanking regions, visualized the direct tandem duplication with a similar, but not identical, genomic organization of the NF1 duplicon copies. Duplication was probably present in the human-chimpanzee-gorilla common ancestor, as demonstrated here by the finding of the duplicated NF1 gene at orthologous chromosome loci. The NF1 intrachromosomal duplication may contribute to the high whole-gene mutation rate by gene conversion, although the functional activity of the NF1 copy remains to be investigated. Detection of the NF1 duplicon by high-resolution FISH may pave the way to filling the gaps in the human genomic sequence of the pericentromeric 17q11.2 region.     

Introgression and characterization of barley yellow dwarf virus resistance from Thinopyrum intermedium into wheat.

Wheatgrasses (species of Agropyron complex) have previously been reported to be resistant to barley yellow dwarf virus (BYDV). To introgress this resistance into wheat, Triticum aestivum x Thinopyrum (Agropyron) intermedium hybrids were advanced through a backcrossing program and reaction to BYDV, as determined by enzyme-linked immunosorbent assay (ELISA), is reported for the first time in backcross populations of wide hybrids between wheat and wheatgrasses. ELISA values revealed highly resistant to highly susceptible segregants in backcrosses. BYDV resistance was expressed in some backcross derivatives. Continued selection, based on cytology and ELISA in each generation, eliminated most of the unwanted wheatgrass chromosomes and produced self-fertile BYDV resistant wheat lines. The BYDV resistant lines with 2n = 42 had normal chromosome pairing similar to wheat, and their F1 hybrids with wheat had two univalents. DNA analyses showed that the source of alien chromatin in these BYDV resistant wheat lines is distinguishable from that in other Th. intermedium derived BYDV resistant wheat lines. Chromosome pairing and restriction fragment length polymorphism analyses indicated that the 42 chromosome resistant Purdue wheat lines are substitution lines in which chromosome 7D was replaced by a chromosome from Th. intermedium that was carrying gene(s) for BYDV resistance.     

A novel 80 kDa human estrogen receptor containing a duplication of exons 6 and 7.

Alterations in the amino acid sequence of the estrogen receptor (ER) have been shown to have dramatic effects on its function. Recently, mutant ERs have been isolated from both clinical samples and established breast cancer cell lines, primarily through the use of the polymerase chain reaction (PCR). All previously reported mutations have given rise to either alterations or truncations of the ER protein. We determined the structure of a novel 80 kDa ER which is expressed in an estrogen independent subclone of the MCF-7 human breast cancer cell line (MCF-7:2A). This 80 kDa ER was initially detected by Western blot analysis using a variety of ER specific antibodies. PCR mapping and partial PCR mediated subcloning of the ER cDNA were used to demonstrate that this protein was an ER containing an in-frame duplication of exons 6 and 7. This type of duplication has not been previously described for any members of the steroid receptor superfamily. Karyotype analysis coupled with fluorescence in situ hybridization (FISH) demonstrated that MCF-7:2A cells contained 4-5 copies of the ER gene in contrast to 2 copies in MCF-7:WS8 cells. The ER gene was localized by FISH analyses in both the MCF-7:WS8 and MCF-7:2A cells on chromosome 6, which is the source of the ER in normal human cells. The relative expression level of 2:1 is consistent with DNA gene dosage analysis. Genomic PCR was then used to demonstrate that the 80 kDa ER mRNA was not derived from the trans-splicing of two ER mRNAs but was the result of a genomic rearrangement in which exons 6 and 7 were duplicated in an in-frame fashion. This variant ER may prove to be useful in elucidating the mechanism of estrogen action in breast cancer cells.     

Expression of recessive Aprt- mutations in mouse CAK cells resulting from chromosome loss and duplication

Karyotypes of recessive mutants at the autosomal adenine phosphoribosyltransferase (Aprt) locus in a clone of the near-diploid mouse CAK cell line have been analyzed. The Aprt located on chromosome 8. One copy of chromosome 8 was morphologically abnormal in the parental clone (CAK-B3-Toyr13) from which Aprt- mutants were isolated. Among 22 mutants, there were ten in which one copy of chromosome 8 had been lost. Four of these were monosomic, and in the others duplication of the remaining homolog had occurred. These findings indicate that newly induced recessive mutations in cultured mammalian cells can be expressed as the result of loss of one chromosome carrying a wild-type allele with or without duplication of the homolog carrying the mutant allele. Loss and duplication would not be detected in cell lines lacking morphologically marked chromosomes.     

Response of Barley Lines with Structural Rearrangements in Chromosomes 5, 6 and 7 to Limited Nitrogen Nutrition

The responses of barley (Hordeum vulgare L.) lines with rebuilt chromosomes 5, 6 and 7 to reduced nitrogen nutrition were evaluated in juvenile growth stages. The material included two series of duplications (D) produced in the short arm of chromosome 6 and of chromosome 7, and in the long arm of chromosome 5 and of chromosome 6; their parental translocation lines (T) - from which analyzed duplications were derived and a standard karyotype cv. Bonus as a control. The translocation lines have break points located in 6_S and 7_S, or 5_L and 6_L. Only the lines with duplicated segments of the short arms of satellited (6 and 7) chromosomes exhibited an improved tolerance to reduced nitrogen supply. No changes relative to cv. Bonus were observed in the T-lines. More tolerant D-lines showed lower stimulation of the root development. Obtained results suggests that the adaptability factors for the low N tolerance at the vegetative growth stage of barley are located in the short arms of 6 and 7 chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Two Cyp19 (P450 aromatase) genes on duplicated zebrafish chromosomes are expressed in ovary or brain

Cytochrome P450 aromatase (Cyp19) is an enzyme catalyzing the synthesis of estrogens, thereby controlling various physiological functions of estrogens. We isolated two cyp19 cDNAs, termed cyp19a and cyp19b, respectively, from zebrafish. These genes are located in linkage groups 18 and 25, respectively. Detailed gene mapping indicated that zebrafish linkage groups 18 and 25 may have arisen from the same ancestral chromosome by a chromosome duplication event. Cyp19a is expressed mainly in the follicular cells lining the vitellogenic oocytes in the ovary during vitellogenesis. Cyp19b is expressed abundantly in the brain, at the hypothalamus and ventral telencephalon, extending to the olfactory bulbs. The expression of duplicated cyp19 genes at two different tissues highlights the evolutionary significance of maintaining two active genes on duplicated zebrafish chromosomes for specific functions in the ovary and the brain.     

Chromosome translocations in breast cancer with breakpoints at 8p12

Unbalanced chromosome translocations with breakpoints around 8p12, resulting in loss of distal 8p, are common in carcinomas. We have mapped the 8p12 breakpoints in three breast cancer cell lines, T-47D, MDA-MB-361, and ZR-75-1, using YACs and PACs between D8S540 and D8S255 by fluorescence in situ hybridization. All three lines had a breakpoint close to D8S505, proximal to HGL. Each breakpoint was distinct, but all were within 0.5 to 1.5 Mb of each other. The T-47D cell line had a straightforward translocation, but in MDA-MB-361 and ZR-75-1 the translocations were accompanied by local rearrangements of surprising complexity. Small regions of 8p from close to the breakpoint were duplicated or amplified as inserts in the attached chromosome fragment. ZR-75-1 also had retained a separate fragment of about 1 Mb, from the region 1 to 3 Mb telomeric to the common breakpoint, that included HGL. This line also had an interstitial deletion several megabases more centromeric. The data suggest that breakpoints on 8p12 are clustered in a small region and show that translocations breaking there may be accompanied by additional rearrangements.     

Duplication and DNA segmental loss in the rice genome: implications for diploidization

* Large-scale duplication events have been recently uncovered in the rice genome, but different interpretations were proposed regarding the extent of the duplications. * Through analysing the 370 Mb genome sequences assembled into 12 chromosomes of Oryza sativa subspecies indica, we detected 10 duplicated blocks on all 12 chromosomes that contained 47% of the total predicted genes. Based on the phylogenetic analysis, we inferred that this was a result of a genome duplication that occurred c. 70 million years ago, supporting the polyploidy origin of the rice genome. In addition, a segmental duplication was also identified involving chromosomes 11 and 12, which occurred c. 5 million years ago. * Following the duplications, there have been large-scale chromosomal rearrangements and deletions. About 30-65% of duplicated genes were lost shortly after the duplications, leading to a rapid diploidization. * Together with other lines of evidence, we propose that polyploidization is still an ongoing process in grasses of polyploidy origins.     

Chromosome anomalies associated with amplification of the adenosine deaminase gene (ADA) in rat hepatoma cells

Two independently selected series of rat hepatoma cell lines resistant to the drug deoxycoformycin (dCF) were analyzed karyotypically. Several forms of homogeneously staining regions (HSRs) were present on metaphase chromosomes of these cells. In some instances HSRs comprised nearly an entire chromosome, which are among the largest chromosomes in the karyotype. Stable resistance to dCF is acquired in rat cells by overproduction of the enzyme adenosine deaminase (ADA) as a result of amplification of ADA gene sequences. We have localized the amplified ADA gene sequences to HSRs on metaphase chromosomes from both series of dCF-resistant cell lines by in situ hybridization. Based upon the number of ADA gene sequences present and the lengths of the HSRs, we have estimated the size of the amplified unit to range from 450 to 1,000 kb.     

杨树基因组中染色体基因丰度分化及EST序列对基因覆盖度的检测(英文)

高等植物基因组中,大部分序列为非表达序列,基因序列所占的比例很小,了解基因在基因组中的分布是研究基因组结构的一个重要方面。在美国能源部资助下,一个毛果杨无性系的基因组测序已经完成并对公众发布。杨树全基因组序列的完成,为我们了解林木基因组中基因的分布提供了一个特例。在本文中,我们利用泊松分析对杨树基因组中基因在各个染色体上的密度进行了检测,结果表明杨树基因组中各条染色体的基因含量存在显著差异。杨树全基因组测序项目揭示现代杨树基因组起源于一次古全基因组复制事件(称为杨柳科基因组复制),所以杨树基因组不同染色体间存在很大的同源复制片段。但是我们的研究显示,杨树基因组中大多数高度同源的染色体上基因的密度与染色体间的同源性没有明显关系,这说明杨柳科全基因组复制事件后,各个高度同源染色体上的基因发生了流失,且基因流失的速率是不一样的。同时本文还对近九万条毛果杨EST序列进行了比对分析,结果显示这些EST序列覆盖的基因仅占杨树基因组中基因总数的16.8%左右。EST测序虽然是发现基因的一个重要手段,但小规模EST测序对基因的覆盖度很低,所以小规模EST测序的应用价值是有限的。     

Asynchronous Duplication of Chromosomes in Cultured Cells of Chinese Hamster

Chromosome duplication (DNA synthesis) was studied in cultured cells of Chinese hamsters by means of autoradiography following thymidine-H³ incorporation. The technique used was to expose an asynchronously dividing population of rapidly growing cells for a 10 minute interval to a medium with thymidine-H³. Cells were then transferred to a medium with excess unlabeled thymidine. The population was sampled at intervals thereafter and studies made of the frequency of labeled interphases and division figures, and the patterns of labeling of specific chromosomes. The average generation time during these experiments was about 14 hours. DNA synthesis occurred during an interval of about 6 hours and stopped 2 to 3 hours before metaphase. After metaphase the chromosomes usually begin duplication again within 5 to 6 hours. Grain counting, to estimate the amount of tritium incorporated after a short contact with thymidine-H³ and at intervals after transfer to a medium with excess unlabeled thymidine, indicated that the intracellular pool of labeled precursors was diluted within less than a minute so that further labeling would not be detected. The chromosomes labeled during the contact period retained their precise pattern of labeling through another duplication cycle and no turnover of DNA or loss of tritium was detectable. Five or 6 chromosomes of the complement have segments typically late in duplication. Two of these are the X and Y chromosomes. The long arm of the X chromosome and the whole Y chromosome are duplicated in the last half of the interval of DNA synthesis. The short arm of the X chromosome in a male strain is duplicated in the first half of the interval. In another strain (female), one X chromosome had the same timing, but the other one was all duplicated in the last half of the period of DNA synthesis. The DNA in the short arms of 2 medium sized chromosomes, as well as most of the DNA in 1 or 2 of the smallest chromosomes of the complement was replicated late. The study has led to the hypothesis that various chromosomes or parts of chromosomes have a genetically controlled sequence in duplication which may have some functional significance.     

A major locus qS12, located in a duplicated segment of chromosome 12, causes spikelet sterility in an indica-japonica rice hybrid

Chromosome segment duplications are integral in genome evolution by providing a source for the origin of new genes. In the rice genome, besides an ancient polyploidy event known in the rice common ancestor, it had been identified that there was a special segmental duplication involving chromosomes 11 and 12, but the biological role of this duplication remains unknown. In this study, by using a set of chromosome segment substitution lines (CSSLs) and near isogenic lines (NILs) derived from the indica cultivar 9311 and japonica cultivar Nipponbare, a major QTL (qS12) resulting in hybrid male sterility was mapped within ~400 kb region adjacent to the special duplicated segment on the short arm of chromosome 12. Compared to the japonica cultivar Nipponbare, the two sides of the qS12 candidate region were inverted in the indica cultivar 9311. Among 47 of the 111 rice genotypes evaluated by molecular markers, the inverted sides were detected, and found completely homologous to indica cultivar 9311. These results suggested that the two inverted sides protect the sequence in the qS12 regions from recombination. On the short-arm of chromosome 12, two QTLs S-e and S25, in addition to qS12, were previously detected as a distinct segregation distortion and pollen semi-sterility loci. We propose these three hybrid sterility loci are the same locus, and the duplicated segment on chromosome 12 may play a prominent role in diversification, i.e., sub-speciation of cultivated rice.     

Evolutionary Dynamics of Recently Duplicated Genes: Selective Constraints on Diverging Paralogs in the <Emphasis Type="Italic">Drosophila pseudoobscura</Emphasis> Genome

Duplicated genes produce genetic variation that can influence the evolution of genomes and phenotypes. In most cases, for a duplicated gene to contribute to evolutionary novelty it must survive the early stages of divergence from its paralog without becoming a pseudogene. I examined the evolutionary dynamics of recently duplicated genes in the Drosophila pseudoobscura genome to understand the factors affecting these early stages of evolution. Paralogs located in closer proximity have higher sequence identity. This suggests that gene conversion occurs more often between duplications in close proximity or that there is more genetic independence between distant paralogs. Partially duplicated genes have a higher likelihood of pseudogenization than completely duplicated genes, but no single factor significantly contributes to the selective constraints on a completely duplicated gene. However, DNA-based duplications and duplications within chromosome arms tend to produce longer duplication tracts than retroposed and inter-arm duplications, and longer duplication tracts are more likely to contain a completely duplicated gene. Therefore, the relative position of paralogs and the mechanism of duplication indirectly affect whether a duplicated gene is retained or pseudogenized. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterisation of mildew resistant wheat-rye substitution lines and identification of an inverted chromosome by fluorescent in situ hybridisation

Seven different mildew resistant wheat lines derived from crosses between triticale and bread wheat were examined by molecular cytogenetics and chromosome C-banding in order to determine their chromosomal composition. Genomic in situ hybridisation (GISH) showed the presence of rye germplasm in all the lines and identified three substitution lines, three double substitution lines and one addition-substitution line. C-banding identified rye chromosomes 1R and 4R in the addition-substitution line, rye chromosomes 1R and 6R in two substitution lines and 1R and 2R in the third line, and rye chromosome 1R in the three substitution lines. Two of the latter lines (7-102 and 7-169) contained a modified form of the chromosome; fluorescent in situ hybridisation (FISH) using five different repetitive DNA-probes showed a pericentric inversion of 1R in both lines. The breakpoints of the 1R inversion were between (1) the 5S rDNA site and the NOR-region on the satellite of the short arm, and (2) between two AAC(5) sites close to the centromere on the long arm. The role of the rye chromosomes in the mildew resistance, the utilisation of the inverted 1R and the significance of the lines in wheat breeding are discussed.