Chung-Mong Chen

The pachytene chromosomes of maize as revealed by fluorescence in situ hybridization with repetitive DNA sequences

Abstract A repetitive DNA sequence, ZmCR2.6c, was isolated from maize based on centromeric sequence CCS1 of the wild grass Brachypodium sylvaticum. ZmCR2.6c is 309 bp in length and shares 65% homology to bases 421–721 of the sorghum centromeric sequence pSau3A9. Fluorescence in situ hybridization (FISH) localized ZmCR2.6c to the primary constrictions of pachytene bivalents and to the stretched regions of MI/AI chromosomes, indicating that ZmCR2.6c is an important part of the centromere. Based on measurements of chromosome lengths and the positions of FISH signals of several cells, a pachytene karyotype was constructed for maize inbred line KYS. The karyotype agrees well with those derived from traditional analyses. Four classes of tandemly repeated sequences were mapped to the karyotype by FISH. Repeats 180 bp long are present in cytologically detectable knobs on 5L, 6S, 6L, 7L, and 9S, as well as at the termini and in the interstitial regions of many chromosomes not reported previously. A most interesting finding is the presence of 180-bp repeats in the NOR-secondary constriction. TR-1 elements co-exist with 180-bp repeats in the knob on 6S and form alone a small cluster in 4L. 26S and 5S rRNA genes are located in the NOR and at 2L.88, respectively. The combination of chromosome length, centromere position, and distribution of the tandem repeats allows all chromosomes to be identified unambiguously. The results presented form an important basis for using FISH for physical mapping and for investigating genome organization in maize.

Two tandemly repeated telomere-associated sequences in Nicotiana plumbaginifolia

Two tandemly repeated telomere-associated sequences, NP3R and NP4R, have been isolated from Nicotiana plumbaginifolia. The length of a repeating unit for NP3R and NP4R is 165 and 180 nucleotides respectively. The abundance of NP3R, NP4R and telomeric repeats is, respectively, 8.4 × 104, 6 × 103 and 1.5 × 106 copies per haploid genome of N. plumbaginifolia. Fluorescence in situ hybridization revealed that NP3R is located at the ends and/or in interstitial regions of all 10 chromosomes and NP4R on the terminal regions of three chromosomes in the haploid genome of N. plumbaginifolia. Sequence homology search revealed that not only are NP3R and NP4R homologous to HRS60 and GRS, respectively, two tandem repeats isolated from N. tabacum, but that NP3R and NP4R are also related to each other, suggesting that they originated from a common ancestral sequence. The role of these repeated sequences in chromosome healing is discussed based on the observation that two to three copies of a telomere-similar sequence were present in each repeating unit of NP3R and NP4R.

A genetic linkage map of Nicotiana plumbaginifolia/ Nicotiana longiflora based on RFLP and RAPD markers

Abstract  We have constructed a genetic linkage map for Nicotiana plumbaginifolia/Nicotiana longiflora (2n= 2x=20), based on the segregation of 69 RFLP and 102 RAPD loci in 99 F2 plants from the cross N. plumbaginifolia×N. longiflora. The map consists of nine major linkage groups, each containing more than nine marker loci, and spans 1062 cM. Twenty of the RFLP markers were mapped previously to Nicotiana sylvestris (2n=2x=24) chromosomes using monosomic alien addition lines. Taxonomically, N. plumbaginifolia and N. sylvestris belong to the same section, namely the Alatae; however, cytogenetic evidence indicates that they are not closely related. Comparison of the distribution of markers common to both maps suggests that genome reorganization has occurred during the evolution of these two species. Evidence is also presented that genome reorganization may be accompanied by gain and loss of specific classes of DNA sequences in their genomes.

Subcellular localization and expression of bamboo mosaic virus satellite RNA-encoded protein

The satellite RNA of bamboo mosaic virus (satBaMV) has a single open reading frame encoding a non-structural protein, P20, which facilitates long-distance movement of satBaMV in BaMV and satBaMV co-infected plants. Immunohistochemistry and immunoelectron microscopy revealed that the P20 protein accumulated in the cytoplasm and nuclei in co-infected cells. P20 and the helper virus coat protein (CP) were highly similar in their subcellular localization, except that aggregates of BaMV virions were not labelled with anti-P20 serum. The BaMV CP protein was fairly abundant in mesophyll cells, whilst P20 was more frequently detected in mesophyll cells and vascular tissues. The expression kinetics of the P20 protein was similar to but slightly earlier than that of CP in co-infected Bambusa oldhamii protoplasts and Nicotiana benthamiana leaves. However, satBaMV-encoded protein levels declined rapidly in the late phase of co-infection. During co-infection, in addition to the intact P20, a low-molecular-mass polypeptide of 16 kDa was identified as a P20 C-terminally truncated product; the possible method of generation of the truncated protein is discussed.

The subtelomeric region of the Arabidopsis thaliana chromosome IIIR contains potential genes and duplicated fragments from other chromosomes

The subtelomere and a portion of the associated telomeric region (together named 3RTAS) of chromosome IIIR from the Arabidopsis thaliana ecotypes Columbia (Col) and Wassilewskija (Ws) were specifically amplified by polymerase chain reaction and subsequently cloned and sequenced. The centromere-proximal portion of 3RTAS from both ecotypes contained two newly identified potential genes, one encoding the chloroplast luminal 19-kDa protein precursor and the other encoding three potential alternatively spliced CCCH-type zinc finger proteins. The telomere-proximal portion of 3RTAS from the Col ecotype contained short duplicated fragments derived from chromosomes I, II, and III, and that from the Ws ecotype contained a duplicated fragment derived from chromosome V. Each duplicated fragment has diverged somewhat in sequence from that of the ectopic template. Small patches of homologous nucleotides were found within the flanking sequences of both the duplicated fragments and the corresponding ectopic template sequences. The structural characteristics of these duplicated fragments suggest that they are filler DNAs captured by non-homologous end joining during double-strand break repair. Our characterization of 3RTAS not only filled up a gap in the chromosome IIIR sequence of A. thaliana but also identified new genes with unknown functions.

Construction and characterization of a Nicotiana plumbaginifolia genomic library in a yeast artificial chromosome

Abstract Large-size DNA of Nicotiana plumbaginifolia was manipulated in solution, concentrated by 2-butanol and cloned into a yeast artificial chromosome, pYACCMC5. A YAC library consisting of 22 000 clones with an average insert size of 190 kb has been constructed. The sizes of YAC inserts in this library ranged from 80–660 kb, indicating that 2-butanol can be used for concentrating large-size DNA with minimum shearing damage. This library represents 84% of the genome of N. plumbaginifolia . Characterization of this library revealed that only 6% of clones in the library contained sequences derived from chloroplast DNA. Some YAC inserts contained relatively long stretches of DNA, 100–180 kb, uninterrupted with repetitive sequences. Furthermore, both ends of most DNA molecules cloned in pYACCMC5 can be reisolated efficiently as recombinant plasmids in Escherichia coli . Approximately one third of the subcloned end fragments contained low- or single-copy DNA sequences. Single-copy end fragments are valuable probes to identify overlapping YAC clones by chromosome walking. The method for concentrating large-size DNA and the vector for YAC cloning described here should be useful in genome research programs. The YAC library presented here will facilitate mapping and cloning of genes from the genome of N. plumbaginifolia , which has been used for over a decade as a model species in plant cell genetics.