Stability yield indices on different sweet corn hybrids based on AMMI analysis.

Currently, sweet corn is considered an important crop due to its high sugar content and low starch content. Important sugars in sweet corn include sucrose, fructose, glucose, and maltose. The purpose of the present study was to use the yield indices of the eight examined sweet corn hybrids and the correlation of the yield indices together. Concentration is important for consumers in terms of yield indices. The research site was located at the Látókép Experimental Station of the University of Debrecen. The small plot experiment had a strip plot design with four replications. The previous crop was sweet corn; the plant density was 64 thousand/ha. The obtained result indicates that Biplot AMMI based on IPCA1 showed that the DB, NO, GS, and GB hybrids had stability and high performance in terms of yield indices. At the same time, fructose and glucose had stable parameters for the hybrids involved in the study. IPCA1 AMMI biplot showed that the ME hybrid had stability and high performance in terms of iron and zinc as well. IPCA2 AMMI biplot showed that DE, GB, and GS hybrids had stability and the highest performance on yield parameters in the scope of the research. Fructose, glucose, and sucrose had stable parameters on hybrids based on IPCA2. The DB and SE hybrids had desirable performance in Lutein and Zeaxanthin based on the biplot. The DE hybrid had a maximum performance on iron and zinc parameters.

Epstein-Barr virus promotes genomic instability in Burkitt's lymphoma.

Epstein-Barr virus (EBV) has been implicated in the pathogenesis of human malignancies but the mechanisms of oncogenesis remain largely unknown. Genomic instability and chromosomal aberrations are hallmarks of malignant transformation. We report that EBV carriage promotes genomic instability in Burkitt's lymphoma (BL). Cytogenetic analysis of EBV- and EBV+ BL lines and their sublines derived by EBV conversion or spontaneous loss of the viral genome revealed a significant increase in dicentric chromosomes, chromosome fragments and chromatid gaps in EBV-carrying cells. Expression of EBV latency I was sufficient for this effect, whereas a stronger effect was observed in cells expressing latency III. Telomere analysis by fluorescent in situ hybridization revealed an overall increase of telomere size and prevalence of telomere fusion and double strand-break fusion in dicentric chromosomes from EBV+ cells. Phosphorylated H2AX, a reporter of DNA damage and ongoing repair, was increased in virus-carrying cells in the absence of exogenous stimuli, whereas efficient activation of DNA repair was observed in both EBV+ and EBV- cells following treatment with etoposide. These findings point to induction of telomere dysfunction and DNA damage as important mechanisms for EBV oncogenesis.

Horizontal transfer of oncogenes by uptake of apoptotic bodies.

Tumor formation involves the accumulation of a series of genetic alterations that are required for malignant growth. In most malignancies, genetic changes can be observed at the chromosomal level as losses or gains of whole or large portions of chromosomes. Here we provide evidence that tumor DNA may be horizontally transferred by the uptake of apoptotic bodies. Phagocytosis of apoptotic bodies derived from H-ras(V12)- and human c-myc-transfected rat fibroblasts resulted in loss of contact inhibition in vitro and a tumorigenic phenotype in vivo. Fluorescence in situ hybridization analysis revealed the presence of rat chromosomes or of rat and mouse fusion chromosomes in the nuclei of the recipient murine cells. The transferred DNA was propagated, provided that the transferred DNA conferred a selective advantage to the cell and that the phagocytotic host cell was p53-negative. These results suggest that lateral transfer of DNA between eukaryotic cells may result in aneuploidy and the accumulation of genetic changes that are necessary for tumor formation.

Similar regions of human chromosome 3 are eliminated from or retained in human/human and human/mouse microcell hybrids during tumor growth in severe combined immunodeficient (SCID) mice.

By passaging microcell hybrids (MCHs) containing human chromosome 3 (chr3) on A9 mouse fibrosarcoma background through severe combined immunodeficient (SCID) mice (elimination test), we have previously defined a 1-Mb-long common eliminated region 1 (CER1) at 3p21.3, a second eliminated region (ER2) at 3p21.1-p14 and a common retained region (CRR) at 3q26-qter. In the present work, chr3 was transferred by microcell fusion into the human nonpapillary renal cell carcinoma line KH39 that contained uniparentally disomic chr3. Four MCHs were generated. Compared with KH39, they developed fewer and smaller tumors, which grew after longer latency periods in SCID mice. The tumors were analyzed in comparison with corresponding MCHs by chr3 arm-specific painting, 19 fluorescent in situ hybridization (FISH) probes, and 27 polymorphic markers. Three MCHs that maintained the intact exogenous chr3 in vitro lost one 3p copy in all 11 tumors. Seven of 11 tumors lost the exogenous 3p, whereas four tumors contained mixed cell populations that lacked either the exogenous or one endogenous KH39 derived 3p. In one MCH the exogenous chr3 showed deletions within CER1 and ER2 already in vitro. It remained essentially unchanged in 8/9 derived tumors. The third, exogenous copy of the 3q26-q27 region (part of CRR) was retained in 16/20 tumors. It can be concluded that the human/human MCH-based elimination test identifies similar eliminated and retained regions on chr3 as the human/murine MCH-based test.

Inactivation of the human fragile histidine triad gene at 3p14.2 in monochromosomal human/mouse microcell hybrid-derived severe combined immunodeficient mouse tumors.

We have previously shown that inoculation of human chromosome 3 (chr3)/A9 mouse fibrosarcoma microcell hybrids (MCHs) into severe combined immunodeficient (SCID) mice was followed by the regular elimination of some 3p regions whereas a 3q region was retained even after prolonged mouse passage. Using this approach, referred to as the elimination test (Et), we have defined a common eliminated region (CER) of approximately 7 cM at 3p21.3 that was absent in all of the 27 tumors generated from five MCHs. Later, CER was reduced to a 1-Mb region, designated as CER1. Another eliminated region (ER2) at 3p21.1-p14.2 was absent in 21 of the 27 tumors. ER2 borders at but does not include the fragile histidine triad (FHIT) gene, considered as a putative tumor suppressor gene. In the present work, two new and two previously studied MCHs, and 13 derived SCID mouse tumors were analyzed by fluorescence in situ hybridization (FISH) chromosome painting and by PCR, using 72 chr3p-specific and 11 chr3q-specific markers. Nine tumors generated from three MCHs that carried cytogenetically normal chr3, remained PCR-positive for all of the chr3 markers tested. Designated as "PCR+" tumors, they were examined by reverse transcription (RT)-PCR, together with four of six previously studied tumors derived from MCH910.7, which carried a del(3)(pter-p21.1), for the expression of 14 human genes: 5 genes within CER1 (LIMD1, CCR1, CCR2, CCR3, CCR5), 5 genes located within regions that were homozygously deleted in a variety of carcinomas (ITGA4L, LUCA1, PTPRG, FHIT, DUTT1), and 4 other genes in chr3p (VHL, MLH1, TGM4, UBE1L). We found that VHL, MLH1, ITGA4L, LIMD1, UBE1L, LUCA1, PTPRG, and DUTT1 were expressed in the MCH lines in vitro and also in the derived SCID tumors. No transcripts that originated from the four CCR genes or from TGM4 could be detected in any of the MCH lines. Alone among the 14 genes examined, FHIT showed a tumor growth-associated change. It was expressed in vitro in five of seven MCH lines. Nine of 13 derived tumors had no FHIT transcript. The remaining 4 expressed a truncated mRNA and a reduced amount of the full-length mRNA. We have previously found that FHIT was deleted at the DNA level in 17 of 21 tumors derived from four MCHs. The remaining 4 of 21 had no FHIT transcript. Our compiled data show that FHIT was either physically or functionally impaired in all 34 of the 34 analyzed tumors. Variants with deleted or down-regulated FHIT have a selective growth advantage.

A 1-Mb PAC contig spanning the common eliminated region 1 (CER1) in microcell hybrid-derived SCID tumors.

We have developed an elimination test to identify chromosomal regions that contain tumor inhibitory genes. Monochromosomal human/mouse microcell hybrids are generated and passaged through SCID mice. Derived tumors are then analyzed for deletions on the transgenomic chromosome. Using this strategy, we have previously identified a 1.6-cM common eliminated region 1 (CER1) on human 3p21. 3. We now report that CER1 contains 14 markers that are deleted in 19 SCID-derived tumors. A 1-Mb PAC contig that spans CER1 was assembled. Five chemokine receptor genes (CCR1, CCR3, CCR2, CCR5, and CCR6) were localized in CER1 in a 225-kb cluster. The lactotransferrin gene (LTF, or lactoferrin, LF), which reportedly has tumor inhibitory activity, also maps to CER1. Our results create a basis for characterization and further functional testing of genes within CER1.

Horizontal transfer of DNA by the uptake of apoptotic bodies.

In this study we have raised the question of whether DNA can be transferred from one cell to another by phagocytosis of apoptotic bodies. We have used integrated copies of the Epstein-Barr virus (EBV) as a marker to follow the fate and expression pattern of apoptotic DNA in the phagocytotic host. Apoptosis was induced in EBV-carrying cell lines by irradiation before cultivation with either human fibroblasts, macrophages, or bovine aortic endothelial cells. Analysis of the expression pattern of EBV-encoded genes was performed by immunofluorescent staining as well as in situ hybridization. Cocultivation of apoptotic bodies from lymphoid cell lines containing integrated but not episomal copies of EBV resulted in expression of the EBV-encoded genes EBER and EBNA1 in the recipient cells at a high frequency. Fluorescence in situ hybridization analysis showed uptake of human chromatin as well as integrated EBV-DNA into the nuclei of bovine aortic endothelial cells. These data show that DNA may be rescued and reused from apoptotic bodies by somatic cells. In addition, our findings suggest that apoptotic bodies derived from EBV-carrying B lymphocytes may serve as the source of viral transfer to cells that lack receptors for the EBV virus in vivo.

Visualization of alternative Epstein-Barr virus expression programs by fluorescent in situ hybridization at the cell level.

Epstein-Barr virus (EBV) transforms human B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). They regularly express six virally encoded nuclear proteins (EBNA1 to EBNA6) and three membrane proteins (LMP1, LMP2A, and LMP2B). In contrast, EBV-carrying Burkitt lymphoma (BL) cells in vivo and derived type I cell lines that maintain the BL phenotype express only EBNA1. During prolonged in vitro culturing, most EBV-carrying BL lines drift toward a more immunoblastic (type II or III) phenotype. Their viral antigen expression is upregulated in parallel. We have used fluorescent in situ hybridization to visualize viral transcripts in type I and III BL lines and LCLs. In type I cells, EBNA1 is encoded by a monocistronic message that originates from the Qp promoter. In type III cells, the EBNA1 transcript is spliced from a giant polycistronic message that originates from one of several alternative Wp or Cp promoters and encodes all six EBNAs. We have obtained a "track" signal with a BamHI W DNA probe that could hybridize with the polycistronic but not with the monocistronic message in two type III BL lines (Namalwa-Cl8 and MUTU III) and three LCLs (LCL IB4-D, LCL-970402, and IARC-171). A BamHI K probe that can hybridize to both the monocistronic and the polycistronic message visualized the same pattern in the type III BLs and the LCLs as the BamHI W probe. A positive signal was obtained with the BamHI K but not the BamHI W probe in the type I BL lines MUTU I and Rael. The RNA track method can thus distinguish between cells that use a type III and those that use a type I program. The former cells hybridize with both the W and the K probes, but the latter cells hybridize with only the K probe. Our findings may open the way for studies of the important but still unanswered question of whether cells with type I latency arise from immunoblasts with a full type III program or are generated by a separate pathway during primary infection.

Stability of a functional murine satellite DNA-based artificial chromosome across mammalian species.

A 60-Mb murine chromosome consisting of murine pericentric satellite DNA and two bands of integrated marker and reporter genes has been generated de novo in a rodent/human hybrid cell line (mM2C1). This prototype mammalian artificial chromosome platform carries a normal centromere, and the expression of its beta-galactosidase reporter gene has remained stable under selection for over 25 months. The novel chromosome was transferred by a modified microcell fusion method to mouse [L-M(TK-)], bovine (P46) and human (EJ30) cell lines. In all cases, the chromosome remained structurally and functionally intact under selection for periods exceeding 3 months from the time of transfer into the new host. In addition, the chromosome was retained in three first-generation tumours when L-M(TK-) cells containing the chromosome were xenografted in severe combined immunodeficiency mice. These data support that a murine satellite DNA-based artificial chromosome can be used as a functional mammalian artificial chromosome and can be maintained in vivo and in cells of heterologous species in vitro.

Restricted expression of Epstein-Barr virus (EBV)-encoded, growth transformation-associated antigens in an EBV- and human herpesvirus type 8-carrying body cavity lymphoma line.

A body cavity lymphoma-derived cell line (BC1), known to carry both Epstein-Barr virus (EBV) and human herpes virus type 8 (HHV-8; or Kaposi's sarcoma-associated herpesvirus, KSHV), was analysed for the expression of EBV-encoded, growth transformation-associated antigens and cellular phenotype by immunofluorescence staining, Western blotting, RT-PCR and flow cytometry. A similar phenotypic analysis was also performed on another body cavity lymphoma line, BCBL1, that is singly infected with HHV-8. Phenotypically, the two lines were closely similar. Although both lines are known to carry rearranged immunoglobulin genes, they were mostly negative for B-cell surface markers. Both expressed the HHV-8-encoded nuclear antigen (LNA1). Similarly to Epstein-Barr nuclear antigen type 1 (EBNA1), LNA1 was associated with the chromatin in interphase nuclei and the mitotic chromosomes in metaphase. It accumulated in a few well-circumscribed nuclear bodies that did not co-localize with EBNA1. BC1 cells expressed EBNA1, LMP2A and EBV-encoded small RNAs but not EBNA2-6, LMP1 and LMP2B. They were thus similar to type I Burkitt's lymphoma cells and latently infected peripheral B-cells. Analysis of the splicing pattern of the EBNA1-encoding message by RT-PCR showed that BC1 cells used the QUK but not the YUK splice, indicating that the mRNA was initiated from Qp and not from Cp or Wp.

Human/mouse microcell hybrid based elimination test reduces the putative tumor suppressor region at 3p21.3 to 1.6 cM.

We have previously identified an approximately 7 cM long common eliminated region (CER), involving the 3p21.3 markers AP20R, D3S966, D3S3559, D3S1029, WI-7947, D3S2354, AFMb362wb9, and D3S32, in human chromosome 3/A9 mouse fibrosarcoma microcell hybrid (MCH) derived SCID mouse tumors. We now report the results of our more detailed analysis on 24 SCID mouse tumors derived from two MCH lines that originally carried intact human chromosomes 3. They were analyzed by fluorescence in situ hybridization (FISH) painting and PCR, using 24 markers covering the region between D3S1611 and D3S13235 at 3p22-p21.2. D3S32 and D3S2354 were regularly eliminated during in vivo tumor growth, whereas the other 22 markers, D3S1611, ACAA, D3S1260, WI-692, AP20R, D3S3521, D3S966, D3S1029, D3S643, WI-2420, MSTI. GNAI2, D3S1235, D3S1298, GLBI, WI-4193, D3S3658, D3S3559, D3S3678, WI-6400, WI-7947, and WI-10865, were regularly retained. We have defined a common eliminated region of approximately 1.6 cM (designated as CER1) inside the 7 cM CER described earlier. CER1 is flanked distally by D3S1029 and proximally by D3S643.

Differential elimination of 3p and retention of 3q segments in human/mouse microcell hybrids during tumor growth.

We have previously found that human chromosome 3 was fragmented in the course of in vivo tumor growth of monochromosomal human/mouse (A9 fibrosarcoma parent) microcell hybrids in SCID mice. Marker analysis of tumor cell lines has identified a regularly eliminated 7 cM segment on 3p21.3 referred to as the common eliminated region (CER). The same region is frequently affected by LOH in a variety of human carcinomas. The present study is a comparative chromosome painting, reverse painting, and PCR marker analysis of microcell hybrids (MCHs) that originally contained an intact chromosome 3 from two alternative donors, during and after four passages in SCID mice. We found regular elimination of 3p in parallel with preferential retention of 3q. In addition to CER on 3p, we can now define a common retained region (CRR) on 3q. It includes eight markers between D3S1282 (3q25-q26) and D3S1265 (3q27-qter) and spans approximately 43 cM. These observations are concordant with the frequent loss of corresponding 3p regions and the frequent retention, with occasional amplification, of 3q in several types of human tumors.

Assignment and ordering of twenty-three unique NotI-linking clones containing expressed genes including the guanosine 5'-monophosphate synthetase gene to human chromosome 3.

Twenty-three unique NotI-linking clones, mainly isolated from the NRL1 library, were mapped and ordered by fluorescence in situ hybridization to human chromosome 3. All these clones were partially sequenced around the NotI sites and thus represent sequence-tagged sites. The EMBL nucleotide database was then searched with sequences from the NotI-linking clones using the FASTA program. This search revealed that the NRL-090 clone (at 3q24) contains the gene encoding human guanosine 5'-monophosphate synthetase (GMPS-PEN). To our knowledge, this is the first localization of this gene. Clone NL1-320 (at 3p21.3) contains a gene encoding arginine tRNA (97.3% identity in 73 bp), while clones NRL-063, NRL-097 and NRL-143 contain expressed sequences with unknown functions. Other clones displayed 60-85% similarities to cDNAs, CpG islands and other genes.

A 3p21.3 region is preferentially eliminated from human chromosome 3/mouse microcell hybrids during tumor growth in SCID mice.

We have previously shown that four markers spanning the 3p24-p21.3 region, THRB, AP20R, D3S1029, and D3S32, were regularly eliminated from three human chromosome 3 (chr3)/mouse microcell hybrids (MCHs) during tumor growth in SCID mice. In an attempt to narrow down the eliminated region, we have studied 22 new SCID mouse tumors derived from 5 MCH lines carrying human chr3. They were analyzed by fluorescence in situ hybridization (FISH), Southern blotting, and polymerase chain reaction (PCR). MCHs that carried human chr1, chr8, chr13, and chr17 were examined as controls. We could identify a common eliminated region (CER) at 3p21.3, bordered distally by D3S1260 and proximally by D3S643/D3F15S2. Eight of 53 chr3-specific PCR markers, AP20R, D3S966, D3S3559, D3S1029, WI-7947, D3S2354, AFMb362wb9, and D3S32. were localized within the CER. This finding is consistent with the notion that a tumor suppressor gene may be located in this area, as suggested by frequent loss of heterozygosity (LOH) within this region observed in several types of solid tumors.

B-CLL cells with unusual properties.

In studies concerning the interaction of B-CLL cells and Epstein-Barr virus (EBV), we encountered one patient whose cells had several unusual properties. In addition to the B-cell markers, the CLL cells expressed the exclusive T-cell markers CD3 and CD8 and carried a translocation t(18,22)(q21;q11), involving the bcl-2 and Ig lambda loci. The patient represents the 4th reported CLL case with this translocation. The CLL cells could be infected and immortalized by the indigenous and by the prototype B958 virus in vitro. The T-cell markers were not detectable on the established lines. In all experiments the immortalized lines originated from the CLL cells. Their preferential emergence over virus-infected normal B cells may be coupled to the high expression of the bcl-2 gene due to the translocation. In spite of the sensitivity of CLL cells to EBV infection in vitro, no EBNA-positive cells were detected in the ex vivo population. In vitro, we could generate cytotoxic function in T-lymphocyte cultures which acted on autologous EBV-infected CLL cells. Therefore we assume that if such cells emerged in vivo they were eliminated by the T-cell response.

Mapping of a new MAP kinase activated protein kinase gene (3PK) to human chromosome band 3p21.2 and ordering of 3PK and two cosmid markers in the 3p22-p21 tumour-suppressor region by two-colour fluorescence in situ hybridization.

A Noti-linking clone NL1-210 (D3S1656) that contains the human MAP kinase activated protein kinase (3PK) gene was localized to 3p21.2 on DAPI-banded and propidium iodide (R-bands)-stained chromosomes by fluorescence in situ hybridization (FISH). For more precise localization of 3PK, two cosmid probes were used as a frame. In order to establish this frame, two Noti-linking clones, NL2-008 (D3S1648) and NL3-003 (D3S3872) were used to screen the cosmid library for locus extension. They mapped to 3p21 and were found to belong to two separate contigs of Noti-jumping and linking clones. Using FISH on DAPI-banded metaphase chromosomes, we have determined the precise localization of cosNL2-008 and cosNL3-003 to 3p21.2-p21.1 and 3p22-p21.3 respectively. The 3PK gene was localized to the 3p21.2 region within this frame by two-colour FISH. The orientation of the probes are tel-D3S3872-3PK-D3S1648-cen.

De novo chromosome formations by large-scale amplification of the centromeric region of mouse chromosomes.

Chromosomes formed de novo which originated from the centromeric region of mouse chromosome 7, have been analysed. These new chromosomes were formed by apparently similar large-scale amplification processes, and are organized into amplicons of approximately 30 Mb. Centromeric satellite DNA was found to be the constant component of all amplicons. Satellite DNA sequences either bordered the large euchromatic amplicons (E-type amplification), or made up the bulk of the constitutive heterochromatic amplicons (H-type amplification). Detailed analysis of a heterochromatic megachromosome formed de novo by an H-type amplification revealed that it is composed of a tandem array of 10-12 large (approximately 30 Mb) amplicons each marked with integrated "foreign' DNA sequences at both ends. Each amplicon is a giant palindrome, consisting of two inverted doublets of approximately 7.5-Mb blocks of satellite DNA. Our results indicate that the building units of the pericentric heterochromatin of mouse chromosomes are approximately 7.5-Mb blocks of satellite DNA flanked by non-satellite sequences. We suggest that the formation de novo of various chromosome segments and chromosomes seen in different cell lines may be the result of large-scale E- and H-type amplification initiated in the pericentric region of chromosomes.

Evidence for a megareplicon covering megabases of centromeric chromosome segments.

We have analysed the replication of the heterochromatic megachromosome that was formed de novo by a large-scale amplification process initiated in the centromeric region of mouse chromosome 7. The megachromosome is organized into amplicons approximately 30 Mb in size, and each amplicon consists of two large inverted repeats delimited by a primary replication initiation site. Our results suggest that these segments represent a higher order replication unit (megareplicon) of the centromeric region of mouse chromosomes. Analysis of the replication of the megareplicons indicates that the pericentric heterochromatin and the centromere of mouse chromosomes begin to replicate early, and that their replication continues through approximately three-quarters of the S-phase. We suggest that a replication-directed mechanism may account for the initiation of large-scale amplification in the centromeric regions of mouse chromosomes, and may also explain the formation of new, stable chromosome segments and chromosomes.