Identification of co-dominant RAPD markers tightly linked to fruit skin color in apple

A simple genetic basis for the red/yellow skincolor polymorphism in apple was verified using DNA markers. Bulked segregant analysis identified one 10-base oligomer that generated different fragments in each of the bulks. After testing the primer in four populations, two fragments were found to be associated with red skin color and another two fragments associated with yellow skin color. Three of the fragments (1160, 1180, and 1230 bp) were partly sequenced and found to share high sequence homology, suggesting these were generated from the same locus. A pair of universal primers were designed to amplify the fragments. In the Rome Beauty x White Angel population, two fragments were associated with red skin color; one fragment designated as A¹ (1160 bp) was from Rome Beauty and another fragment (A², 1180 bp) was from White Angel. Progeny possessing both fragments, or either one, had red fruit. Both parents displayed an alternate fragment, a¹ (1230 bp), associated with yellowskinned fruit. In three other crosses tested, only fragment A¹ co-segregated with red skin color; two fragments, a¹ and a² (1230 bp and 1320 bp), were associated with yellow skin color. Our results are consistent with the hypothesis that the red/yellow dimorphism is controlled by a monogenic system with the presence of the red anthocyanin pigmentation being dominant. There was no indication that other modifier genes could reverse the effect of the locus (R _f ) linked to the markers. Examination of amplification products in 56 apple cultivars and advanced breeding selections demonstrated that the universal primers could be used to correctly predict fruit skin color in most cases.     

The spectra of base substitutions induced by the impCAB, mucAB and umuDC error-prone DNA repair operons differ following exposure to methyl methanesulfonate

We have used the lacZ reversion assay to study the mutation spectra induced by the Escherichia coli chromosomal umuDC operon and of its two plasmid-borne analogues impCAB and mucAB following exposure of cells to UV light and methyl methane-sulfonate (MMS). We have shown that the impCAB, mucAB and umuDC operons all produce a similar response to UV light which results almost exclusively in AT GC transitions. However, we found that the three operons produced different responses to alkylating agents. We found that with MMS the chromosomal umuDC operon produced almost exclusively AT GC transitions, whilst both mucAB and impCAB produced predominantly transversions. In the case of the impCAB operon the mutation spectrum contained more AT TA than GC TA transversions; this balance was reversed with mucAB. The effect of the copy number of the error-prone DNA repair operons upon the mutagenic spectra was also studied. The results obtained suggest that the copy number of the imp operon does not greatly affect the specificity of base substitutions observed. However, an increase in the copy number of the umuDC operon greatly affected the specificity of base substitution, such that virtually no transitions were produced and the spectrum was dominated by GC/AT TA transversions. It appears that the three error-prone DNA repair operons impCAB, mucAB and umuDC, despite showing strong structural and functional homologies, can display major differences in the spectrum of base changes induced during mutagenesis. We propose that the type of misincorporation/chain extension which DNA polymerase III is allowed to synthesize on a damaged DNA template is extremely sensitive to both the amount and type of error-prone repair proteins present. The modulation of these events by the different proteins can result in widely different mutagenic changes in the repaired DNA.     

The Use of Competitive PCR Mimic to Evaluate a Limulus Lambda Phage Genomic DNA Library

1. A phage genomic DNA library for Limulus (L.) polyphemus brain was constructed using the GEM-12 vector and the host strain KW251.2. The primary library contained approximately 1.275 × 10⁶ independent clones, increasing upon amplfication to 6.66 × 10⁹ pfu/ml in a total volume of 58 ml.3. A total of 28 clones was randomly chosen for a determination of the average size of inserts in the library. All clones contained inserts and the average size was 14.9 kb, ranging from 11.7 to 28.0 kb. The library provides a 10-fold equivalent of the L. polyphemus genome.4. A new approach for evaluating a genomic DNA library was developed, in which competitive PCR MIMIC was employed to determine the target gene copy number in both constructed library and brain genomic DNA. The putative protein kinase C (PKC) was selected as the target gene because its partial sequence of cDNA was recently cloned from L. polyphemus brain in our laboratory (Cao et al., 1998). A 419-bp fragment of nonhomologous sequence derived from putative PKC and a 306-bp fragment from plasmid pUC 18 were generated for use as target and competitor in PCR MIMIC, respectively.5. Within the genomic library DNA, a 0.8 value was obtained for the copy number of the putative PKC gene that was detected in 0.1 amol of one equivalent L. polyphemus genome in terms of the average recombinant molecular weight. In the genomic DNA, a single copy of putative PKC was found in 0.1 amol of one coverage for the L. polyphemus genome. Thus, it was implied that nearly 80% genetic resource was incorporated into the library. This percentage was termed the incorporation rate.6. Based on these findings, we suggest that the incorporation rate is an essential factor for evaluating genomic libraries, particularly, when using partial digestion with restriction enzymes for library construction.     

Variable methylation and differential replication of genomic DNA in cultured carrot root expiants during growth induction as influenced by hormonal treatments

Summary The methylation and amplification pattern of genomic DNA of carrot root expiants (Daucus carota L.) undergoes transitory changes during the cultural cycle. A high degree of variation was observed as early as 36 h after the incubation of fresh expiants in the nutrient medium and, depending on the hormonal treatment significant modifications occurred during 14 days of culture. Proliferative tissue conditioned by kinetin showed an extensive reduction in DNA methylation. Changes in the DNA amplification pattern were not necessarily linked to methylation.     

Assignment of anonymous DNA probes to specific intervals of human chromosomes 16 and X

Summary Anonymous DNA probes mapping to human chromosome 16 and the distal region of the human X chromosome were isolated from a genomic library constructed using lambda EMBL3 and DNA from a mouse/human hybrid. The hybrid cell contained a der(16)t(X;16)(q26;q24) as the only human chromosome. Fifty clones were isolated using total human DNA as a hybridisation probe. Forty six clones contained single copy DNA in addition to the repetitive DNA. Pre-reassociation with sonicated human DNA was used to map these clones by a combination of Southern blot analysis of a hybrid cell panel containing fragments of chromosomes 16 and X and in situ hybridisation. One clone mapped to 16pter 16p13.11, one clone to 16p13.316p13.11, four clones to 16p13.316p13.13, two clones to 16p13.1316p13.11, one clone to 16p13.11, seven clones to 16p13.1116q12 or 16q13, four clones to 16q12 or 16q13, three clones to 16q1316q22.1, four clones to 16q22.10516q24, and nineteen clones to Xq26Xqter. Two clones mapping to 16p13 detected RFLPs. VK5 (D16S94) detected an MspI RFLP, PIC 0.37. VK20 (D16S96) detected a TaqI RFLP, PIC 0.37 and two MspI RFLPs, PIC 0.30 and 0.50. The adult polycystic kidney disease locus (PKD1) has also been assigned to 16p13. The RFLPs described will be of use for genetic counselling and in the isolation of the PKD1 gene. Similarly, the X clones may be used to isolate RFLPs for genetic counselling and the isolation of genes for the many diseases that map to Xq26qter.     

Effects of supplied cinnamic acids and biosynthetic intermediates on the anthocyanins accumulated by wild carrot suspension cultures

Anthocyanins isolated and characterized from the wild carrot suspension cultures used here were 3-O--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D<-galactopyranosylcyanidin (1), 3-O-[-D- xylopyranosyl-(12)--D-galactopyranosyl]cyanidin (2), 3-O-(6-O-sinapoyl)--D-glucopyranosyl-(16)-[-D- xylopyranosyl-(12)-]-D-galactopyranos ylcyanidin (3), 3-O-(6-O-feruoyl)--D-glucopyranosyl-(16)-[- D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (4), 3-O-(6-O-coumaroyl)--D-glucopyranosyl-(16)- [-D-xylopyranosyl-(12)-]-D-galactopyrano sylcyanidin (5), 3-O-[6-O-(3,4,5-trimethoxycinnamoyl)]-- D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (6), 3-O-[6-O-(3,4-dime- thoxycinnamoyl)]--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (7), 3-O-[(6-O-sinapoyl)--D-glucopyranosyl-(16)--D-galactopyranosyl]cyanidin (8), and 3-O-(-D-galactopyranosyl)cyanidin (9). Except when cinnamic acids were provided in the culture medium, the major anthocyanin present in the two clones examined was 2. When the naturally occurring and some non-naturally occurring cinnamic acids were provided individually in the medium, 1 and 2 were minor components and the anthocyanin acylated with the supplied cinnamic acid, namely 3, 4, 5, 6, or 7 was the major anthocyanin present in the tissue. When caffeic acid was provided the major anthocyanin in the tissue was 4, thereby suggesting that the caffeic acid was methylated before its use in anthocyanin biosynthesis. Other cinnamic acids supplied had limited effects on the anthocyanins accumulated and appeared not to result in the accumulation of new anthocyanins by the tissue. Thus the tissue can use some but not all analogues of sinapic acid to acylate anthocyanins. Additional anthocyanins were detected in extracts of the wild carrot tissue cultures using mass spectrometry (both MS/MS and HPLC/MS). The additional compounds detected have also been found in cultures of black carrot, an Afghan cultivar of Daucus carota ssp. sativa and the flowers of wild carrot giving no evidence for qualitative differences in the anthocyanins synthesized by subspecies, cell cultures from subspecies, or clones from cell cultures. There are major differences in the amounts of individual anthocyanins found in cultures from different subspecies and in different clones from cell cultures. Here anthocyanins without acyl groups were usually found in the tissues and their accumulation is discussed. On the basis of the structures of the isolated anthocyanins, a likely pathway from cyanidin to the accumulated anthocyanins is proposed and discussed.Abbreviations Sin sinapoyl - Fer feruoyl - 4-Coum. 4-coumaroyl - 3,4-MeO₂Cin 3,4-dimethoxyeinnamoyl - 3,4,5-MeO₃Cin 3,4,5-trimethoxycinnamoyl - Cya cyanidin     

Physiological aspects of genome variability in tissue culture. II. Growth phase-dependent quantitative variability of repetitive BstNI fragments of primary cultures of Daucus carota L.

Systematic investigations on the occurrence of differential DNA replication in carrot cultures, expressed at the total genome level, were performed. The genome of Daucus carota L. could be characterized by a pattern of repetitive BstNI fragments that was independent of tissue specificity or cultivar differences. Characterization of the genomic DNA of the secondary phloem of carrot roots, in comparison to the DNA of the induced primary cultures at different growth phases, revealed dramatic differences in the copy number of the repetitive fragments. Highly proliferative tissue showed extensive reduction in the proportion of repetitive sequences in the genome in all of the 37 investigated variants. In contrast, during subsequent transition to stationary growth the repetitive fragments re-amplified. The results suggest that the quantitative genome organisation was involved in the regulation of the growth potential of cells. A hypothesis is discussed suggesting a determining influence of the observed differential DNA replication on cell-cycle rates and the cell program of proliferative tissue by structural and positioning effects on DNA loops. To study the causality of somaclonal variation, research on the relationship between physiological genome variability and the induction of heritable changes is recommended.     

Transgene structures in T-DNA-inserted rice plants

T-DNA is commonly used for delivery of foreign genes and as an insertional mutagen. Although ample information exists regarding T-DNA organization in dicotyledonous plants, little is known about the monocot rice. Here, we investigated the structure of T-DNA in a large number of transgenic rice plants. Analysis of the T-DNA borders revealed that more than half of the right ends were at the cleavage site, whereas the left ends were not conserved and were deleted up to 180 bp from the left border (LB) cleavage site. Three types of junctions were found between T-DNA and genomic DNA. In the first, up to seven nucleotide overlaps were present. The frequency of this type was much higher in the LB region than at the right border (RB). In the second type, which was more frequent in RB, the link was direct, without any overlaps or filler DNA. Finally, the third type showed filler DNA between T-DNA and the plant sequences. Out of 171 samples examined, 77 carried the vector backbone sequence, with the majority caused by the failure of T-strand termination at LB. However, a significant portion also resulted from co-integration of T-DNA and the vector backbone to a single locus. Most linkages between T-DNA and the vector backbone were formed between two 3 ends or two 5 ends of the transferred DNAs. The 3 ends were mostly linked through 3–6 bp of the complementing sequence, whereas the 5 ends were linked through either precise junctions or imprecise junctions with filler DNA.     

Maintenance of the induced hypomethylated state of tobacco nuclear repetitive DNA sequences in the course of protoplast and plant regeneration

Previously, we established experimental conditions allowing us to induce hypomethylation of tandem arrays of highly repetitive DNA sequences in the Nicotiana tabacum L. nuclear genome (M. Bezdk et al. 1991, Planta 184, 487–490). In this paper, we demonstrate that loci containing the highly repetitive sequences of the HRS60 family can maintain the induced hypomethylated state through protoplast regeneration, non-differentiated callus growth, and plant regeneration. The hypomethylation, induced with 5-azacytidine and monitored on these sequences, did not substantially alter the capacity of calli to produce plants. It can be concluded that, in contradistinction of multiple copies of transgenes or ectopic genes which are usually recognized as methylation targets, endogenous tandem repeats, such as the HRS60, present at 10⁵ copies in the genome, can escape de-novo methylation.Abbreviation AzaC 5-azacytidine This project was supported by the Grant Agency of the Academy of Sciences of the Czech Republic. We thank Ms. Libue Jedliková, Ms. Hana Suchánková, and Ms. Emilie Koudelková for technical assistance.     

Integration of DNA copy number alterations and transcriptional expression analysis in human gastric cancer

Background

Genomic instability with frequent DNA copy number alterations is one of the key hallmarks of carcinogenesis. The chromosomal regions with frequent DNA copy number gain and loss in human gastric cancer are still poorly defined. It remains unknown how the DNA copy number variations contributes to the changes of gene expression profiles, especially on the global level.

Principal Findings

We analyzed DNA copy number alterations in 64 human gastric cancer samples and 8 gastric cancer cell lines using bacterial artificial chromosome (BAC) arrays based comparative genomic hybridization (aCGH). Statistical analysis was applied to correlate previously published gene expression data obtained from cDNA microarrays with corresponding DNA copy number variation data to identify candidate oncogenes and tumor suppressor genes. We found that gastric cancer samples showed recurrent DNA copy number variations, including gains at 5p, 8q, 20p, 20q, and losses at 4q, 9p, 18q, 21q. The most frequent regions of amplification were 20q12 (7/72), 20q12–20q13.1 (12/72), 20q13.1–20q13.2 (11/72) and 20q13.2–20q13.3 (6/72). The most frequent deleted region was 9p21 (8/72). Correlating gene expression array data with aCGH identified 321 candidate oncogenes, which were overexpressed and showed frequent DNA copy number gains; and 12 candidate tumor suppressor genes which were down-regulated and showed frequent DNA copy number losses in human gastric cancers. Three networks of significantly expressed genes in gastric cancer samples were identified by ingenuity pathway analysis.

Conclusions

This study provides insight into DNA copy number variations and their contribution to altered gene expression profiles during human gastric cancer development. It provides novel candidate driver oncogenes or tumor suppressor genes for human gastric cancer, useful pathway maps for the future understanding of the molecular pathogenesis of this malignancy, and the construction of new therapeutic targets.     

Physiological aspects of genome variability in tissue culture. I. Growth phase-dependent differential DNA methylation of the carrot genome (Daucus carota L.) during primary culture

Investigations were performed on growth phase-dependent EcoRII site-specific DNA methylation of the carrot genome during primary culture to elucidate physiological aspects of genome DNA variability in tissue culture. While DNA methylation of the root cambium and the secondary phloem and petioles of carrot leaves were strikingly different, the methylation level of the secondary phloem seemed to be independent of cultivar origin, the age of the plants and the extent of secondary root growth. As was shown earlier a change in the differentiated state of the secondary phloem by tissue culture leads to changes in genome modification. Whereas de novo methylation was observed during the first 2 weeks of growth initiation, the results presented demonstrate genome de-methylation during the transition to stationary growth indicating differential nome methylation during different phases of culture. The presence of kinetin in the nutrient medium of the primary culture was found to be antagonistic to changes in genome modification in general. De novo methylation and subsequent de-methylation of the carrot genome are discussed as gross changes obviously essential to molecular genome differentiation during tissue culture.     

Somatic embryogenesis,plant regeneration and somaclonal variation in barley

In vitro culture of immature embryo and young leaf tissues was carried out with five cultivars of barley, Hordeum vulgare. Two cultivars (Albacete and Porthos) responded poorly from both types of explants, while the three others (Dissa, Golden Promise and Ingrid) produced a high frequency of embryogenic callus from these explants (25–60%). For Dissa and Ingrid, young leaf explants were slightly better than immature embryo explants for embryogenic callus induction, while immature embryo cultures of Golden Promise responded better than young leaf explants. Thus, there appears to be a significant genotype × explant interaction in the initiation of embryogenic callus in barley.Some phenotypic variants were detected among the regenerated plants of Golden Promise and Ingrid, most originating by epigenetic changes. Only in one case was the variant phenotype heritable, probably due to a mutation in the chloroplast DNA. Mitotic alteractions were not detected. Consequently, somaclonal variation did not appear to be a very frequent event in plants regenerated from 1- to 6- month-old cultures of barley.     

Cloning of the dnaB gene of Escherichia coli: the dnaB gene of groPB534 and groPB612 and the replication of phage lambda

Summary Fragments of the E. coli chromosome that carry the dnaB groPB534 or groPB612 alleles have been cloned into a cosmid vector. The resulting recombinant plasmids contained the genes uvrA, groP (B534 or B612), and lexA. Further subcloning into high copy number plasmids, during which the uvrA and lexA genes were removed successively, yielded a groPB534 and groPB612 DNA fragment of about 2.4 kb each. Both fragments contained an overlapping 1.8 kb segment of DNA in which the sites of all restriction enzymes tested were identical. The size of these dnaB gene fragments were further delimited by deletion analysis.In E. coli groPB534 in which wild-type and A mutants do not replicate (Georgopoulos and Herskowitz 1971) phage replication is rescued if the strain contains the groPB534 gene on high copy number plasmids. On the contrary, in E. coli groPB612, which is temperature-sensitive for its groP character, replication of and A is abolished at 30° C if the strain contains the groPB612 recombinant plasmid. On the other hand, replication of B remains unaffected whether or not the groP strains harbor the isogenic dnaB gene-containing plasmid. The results suggest that within the cell not only the quality but also the relative amounts of dnaB and P protein are crucial for phage replication.     

Isolation and characterization of an α-satellite repeated sequence from human chromosome 22

We constructed a library in IL47.1 with DNA isolated from flow-sorted human chromosome 22. Over 50% of the recombinants contained the same highly repetitive sequence. When this sequence was used to probe Southern blots of EcoRI-digested genomic DNA, a ladder of bands with increments of about 170 bp was observed. This sequence comigrates with satellite III in Ag⁺/Cs₂SO₄ gradients and may account for at least part of the 170 bp Hae III ladder seen in isolated satellite III DNA. Partial sequence analysis revealed homology to the 171 bp monomeric repeat unit of -R1-DNA and the X specific -satellite consensus sequence. After low stringency in situ hybridization, silver grains were found over the centromeres of a number of chromosomes. Under high stringency conditions, however, the labeling was concentrated over the centromeric region of chromosome 22. This localization was confirmed using DNA from a panel of human/hamster cell lines which showed that the homologous 2.1 and 2.8 kb EcoR1 restriction fragments were chromosome 22 specific. These clones therefore contain chromosome 22 derived -satellite sequences analogous to other chromosome-specific satellite sequences described previously.     

Isolation and characterization of a multienzyme complex containing DNA replicative enzymes from mitochondria ofS. cerevisiae

A 40 S multienzyme complex containing mtDNA polymerase was isolated from mitochondria ofS. cerevisiae by density gradient centrifugation and by gel filtration chromatography. Besides DNA polymerase, RNA polymerase, primase, 35 exonuclease and an ATPase activities were found to be associated with it. The presence of some of these enzymes were confirmed by Western blot. This high molecular weight multienzyme complex containing DNA has most of the attributes of a putative replisome.Abbreviations BCIP 5 bromo 4 chloro 3 indolyl phosphate - mtDNA mitochondrial DNA - NBT Nitroblue tetrazolium - PBS phosphate buffered saline     

Mitochondrial DNA rearrangements in somatic hybrids of Solanum tuberosum and Solanum brevidens

Summary Thirty somatic hybrids between Solanum tuberosum and Solanum brevidens were analysed for mitochondrial and chloroplast genome rearrangements. In all cases, the chloroplast genomes were inherited from one of the parental protoplast populations. No chloroplast DNA alterations were evident but a range of mitochondrial DNA alterations, from zero to extensive intra- and inter-molecular recombinations, were found. Such recombinations involved specific recombination hot spots in the mitochondrial genome. Not all hybrids regenerated from a common callus possessed identical mitochondrial genomes, suggesting that sorting out of mitochondrial populations in the callus may have been incomplete at the plant regeneration stage. Sorting out of organelles in planta was not observed.     

Identification of a multigene family for small heat shock proteins in soybean and physical characterization of one individual gene coding region

When soybean seedlings are tranferred from 28 to 40 ° C, a heat shock (hs) response is elicited. This is characterized by the synthesis of a new set of proteins (hs-proteins) and by cessation of normal protein synthesis (8). At the level of poly(A)mRNA, a new class of highly abundant RNAs appears which encodes a group of hs-proteins in the low molecular weight range of 15–18 kD (11). The classification of these proteins/genes into several sub-classes is based on a complex sequence relationship for class I protein/genes.This was confirmed by both the complexity and the similarity of southern blot hybridization patterns of genomic DNA digests with class I cDNA-probes. Genomic DNA clones (obtained from -libraries by screening with cDNA-probes) for the class I gene 1968 showed cross hybridization with all other class I cDNA-probes. Higher specificity of gene/protein correlation was obtained by variation of hybridization criteria. The specificity of cDNA clone 1968 for the genomic DNA clone hs68-7 was demonstrated by thermal stability of hybridization at 55 ° C and 65 ° C in 50% formamide compared to other cross-reacting probes. The correlation of clone 1968 with a specific hs-protein was obtained by temperature dependent release of hybrid selected hs-mRNAs at 50, 60, 70 and 85 ° C followed byin vitro translation and two-dimensional gel analysis. The coding regions of hs-genes on genomic DNA clones were mapped by R-loop formation. The position of R-loops was mapped relative to certain restriction sites on subclones of hs68-7 DNA. The polarity of hs-genes was determined by attaching X174RF-DNA labels to the 3 poly(A)-tails of the mRNAs of R-loops.     

Genotypic control of pollen plant formation in Nicotiana tabacum L.

Summary Tobacco plants (Nicotiana tabacum L.) of four varieties (Badischer Burley, White Burley, Techne, Kupchunos) were raised at different temperatures and daylengths and the effect of genotype on embryogenic pollen grain formation in situ and on pollen plant formation in anther and pollen cultures from these plants was studied. Genotype controlled embryogenic pollen grain and pollen plant formation by defining productivity under standard growth conditions (long days at 24 °C). Kupchunos was the most productive variety, followed by White Burley, Techne, and Badischer Burley. Furthermore, genotype defined which environmental factor was able to affect embryogenic pollen grain and pollen plant formation and also to which degree. In anther cultures, in addition to these effects, genotype controlled the formation of (an) inhibitory substance(s) in the anther wall in interaction with the plant growth conditions. In Badischer Burley and Techne, inhibitor action could be prevented by isolation of the pollen after one week of anther culture. Finally, direct pollen cultures in Badischer Burley and Techne produced embryos were only when the pollen was isolated from nearly mature anthers, while in White Burley and Kupchunos, embryos also produced at earlier stages and at higher yields. This indicated that genotype controls the time when the embryogenic pollen grains become ready to divide. The results are discussed in relation to strategies to overcome recalcitrance of species and genotypes.     

Primary callus as source of totipotent barley (Hordeum vulgare L.) protoplasts

Summary Primary callus of barley (Hordeum vulgare L.) derived from scutella (cv. Dissa) and anthers (cv. Igri) was used for protoplast isolation and plant regeneration. The protoplasts were embedded in agarose and cultured with nurse cells. The plating efficiency varied from 0.1% to 0.7%. Shoots regenerated from the developing callus. Plantlets were transferred to soil and cultivated in the greenhouse three to five months after protoplast isolation. All plants were normal in morphology, and most of them flowered and set seeds.     

Development of a sequence-tagged site for the centromere of chromosome 10: its use in cytogenetic and physical mapping

We sequenced the alphoid centromere probe p10RP8 (D10Z1), aligned it to three published consensus sequences, and developed a sequence-tagged site (STS), sJRH-2, based upon oligonucleotide primers having two 3 mismatches with these consensus sequences. Polymerase chain reaction (PCR) amplification using genomic DNA from a somatic cell hybrid panel representing all human chromosomes demonstrated amplification from only those cell lines containing chromosome 10. Fluorescence in situ hybridization of the amplified product demonstrated intense and specific hybridization of the PCR product to 10p11.1-q11.1. A human genomic yeast artificial chromosome (YAC) library was screened using the sJRH-2 PCR assay, and five clones were identified. Sequence analysis of one chimeric clone (consisting of DNA segments derived from chromosomes 5p and 10cen) confirmed specificity of the STS for the centromere of chromosome 10. sJRH-2 provides a convenient cytogenetic marker for chromosome 10, which will also be useful for physical mapping of the pericentromeric region of chromosome 10, a region that harbors the gene(s) for three forms of multiple endocrine neoplasia (types 2A, 2B, and familial medullary thyroid carcinoma). The GenBank accession number for the p10RP8 sequence is X63622.