Article Figures & Data
Figures
- Fig. 1.
Array comparative genomic hybridization analysis of normal versus female. A, representative genomic profile of an array comparative genomic hybridization using normal male versus normal female DNAs. Six simultaneous hybridizations of normal male versus normal male were performed to define the normal variation in log2 ratio (log2 cy3/cy5). In the control experiment, >95% of the measured fluorescence log2 ratio values of each spot (2 × 1966 clones) ranged from +0.2 to −0.2 (data not shown). The thresholds for the log2 ratio of gains and loss were therefore set at log2 ratio of +0.2 and −0.2, respectively. Array data are plotted as the mean log2 ratio of duplicate spots for each clone. Vertical lines show the threshold for the log2 ratio of gains and loss. The log2 ratio for each of the bacterial artificial chromosome clone is plotted as a function of its genome location, with chromosome 1 to the left and X to the right; for each chromosome the order is short-arm telomeric to long-arm telomeric. B, normalized log2 ratio for the changes in copy number of X chromosome. The normal male DNA was used as reference for all hybridizations. Array hybridizations were performed with the test genomic DNA from a normal male (1 × chromosome), a normal female (2 × chromosomes), and three cell lines containing three, four, and five copies of the X chromosome. Each plot stands for the mean value of all normalized fluorescence ratio of 57 clones from the X chromosome. The ratio on each of the X chromosome clone was normalized by the mean fluorescence intensity ratio of autosomal chromosome clones. We defined the fluorescence intensity ratio of array-hybridization with normal male versus normal male as 0. Each plot was then computed on the basis of the normalized value. The line represents the linear regression through all of the data with a slope of 0.51 and an intercept of 0.72.
- Fig. 2.
Genomic profiles of array comparative genomic hybridization. A, the representative genomic profile of array comparative genomic hybridization with Karpas 1718. The bacterial artificial chromosomes are ordered by position in the genome beginning at the 1p telomere and ending at the Xq telomere. The black arrow above the graph indicates high-level amplification (defined as log2 ratio >1). B, detailed genomic profiles of chromosome 13 in the three cell lines (Karpas 1718, Rec1, and OCI-Ly7) and one diffuse large B cell lymphoma patient (D778). The log2 ratio for each of the 68 bacterial artificial chromosomes and P-1 derived artificial chromosome clones is plotted as a function of its genome location, with chromosome 13q-centromere to the left and 13q-telomere to the right. ····, show the threshold for gains and loss. Bold arrows indicate high-level amplification (defined as log2 ratio >1) and thin arrows moderate-level amplification (0.2 > log2 ratio >1). Karpas 1718 shows a wide region of amplification extending over >50-Mb of chromosome 13q. Furthermore, high-level amplification in Karpas 1718 is observed from 13q22.2 to 13q31.3, with 13q31.3 in particular showing the highest amplification (log2 ratio >2). In the same manner, high-level amplification of OCI-Ly7 and Rec1 are shown at 13q31.3. Rec1 also shows wide loss in the vicinity of 13q31.3. The patient sample (D778) shows a wide region of amplification at 13q21.2–13q31.3 and 13q33.3-qter, with 13q31.1-q31.3 in particular indicating high-level amplification.
- Fig. 3.
Fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH) analysis of cell lines with or without amplification at 13q31-q32. CGH results for four cell lines (Karpas 1718, Rec1, OCI-Ly4, and Jurkat) are shown. The lines to the right (green) and to the left (red) of each chromosome indicate the region gained or lost, respectively. Representative results of metaphase FISH with bacterial artificial chromosome, RP11–487A2 are shown on the right side of each panel. Chromosome 13 examined by CGH is also shown beside each ideogram. Three B-cell lymphoma cell lines, Karpas 1718 (A), Rec1 (B), and OCI-Ly4 (C) show amplification at 13q31-q32, but Jurkat (D) does not show one. FISH analysis shows amplification in >15 copies in the three B-cell lines, but no amplification in Jurkat. Each metaphase chromosome was counterstained by 4′,6-diamidino-2-phenylindole.
- Fig. 4.
Analysis of 13q31-q32 by a combination of array comparative genomic hybridization (CGH) and interphase fluorescent in situ hybridization (FISH). A, summarized data of DNA sequence copy numbers in three cell lines (Karpas 1718, OCI-Ly4, and Rec1) determined by interphase FISH using 19 bacterial artificial chromosome (BAC) clones of 13q31.3, including a new BAC, RP11–93M14, that was not used for array CGH. Ten interphase cells were analyzed and the average copy numbers of the BAC clone signals were counted for each cell line. The vertical line indicates the copy number and the horizontal dotted line indicates normal two copies.
shows the common region of gain in copy number, which extended fromRP11–29C8 to RP11–93M14. The positions of STS markers and all BAC clones were confirmed from information archived by Ensembl Genome Data Resource.
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The underlined BAC clones were used for FISH and array CGH. The thin arrow indicates the GPC5 gene loci. B, summarized data of array CGH analysis of three cell lines (Rec1, Karpas 1718, and OCI-Ly7) and one DLBCL patient (D778). The vertical line shows log2 ratio. ···· show the threshold for gain and loss set at log2 ratios of +0.2 and −0.2, respectively.
shows the common region of high-level amplification (log2 ratio >1) in the three cell lines, which is extended from RP11–360A9 to RP11–481A22. - Fig. 5.
Northern blot analysis of the candidate gene for 13q31-q32 amplification. Northern hybridization was performed against 6 kinds of RNAs comprising human placenta (lane 1), 3 B-cell lymphoma cell lines (lane 2, Rec1; lane 3, Karpas 1718; and lane 4, OCI-Ly4) with amplification at 13q31-q32 and 2 T-cell lymphoma cell lines (lane 5, Jurkat and lane 6, ATN-1) without amplification. Representative and characteristic expression patterns of 8 of 30 ESTs and GPC5 are shown. Expression of GPC5 and BI481522 was not significantly different, whereas LOC160824, AF339828, BC040320, AF339802, LOC121734, AA705439, and N49442 showed clearly different patterns of expression. In particular, the expression of AF339828 and BC040320, which showed similar patterns of hybridization, demonstrates concordance with the gain in copy number at 13q31-q32.
- Fig. 6.
Expression study of GPC5 and BC040320. Amplification status at 13q31-q32 in each of the cell lines and diffuse large B-cell lymphoma (DLBCL) patients examined by conventional comparative genomic hybridization is indicated above names of the samples. A, expression pattern of GPC5 and BC040320 in cell lines and DLBCL patients with or without amplification at 13q31-q32. Expression of GPC5 in five cell lines and two DLBCL patients with amplification at 13q31-q32 is not significantly different from that of the other cell lines and patients without amplification. BC040320 is expressed in cell lines with amplification at 13q31-q32 (lanes 1–5) and at much lower levels in cell lines without amplification (lanes 6–8). In the same manner, BC040320 is strongly expressed in DLBCL patients with amplification at 13q31-q32 (lanes 9 and 10), but very weakly in cell lines without amplification (lanes 11 and 12). B, expression pattern of GPC5 and BC040320 in multiple cell lines with hematopoietic malignancies. Some cell lines (lanes 9, 11, and 12) with amplification at 13q31-q32 show weak signals when compared with the two cell lines (lanes 1 and 2) with high-level amplification. Expression of GPC5 shows very weak signals with some variations but without significant differences. AML, acute myeloid leukemia cell line. MM, multiple myeloma cell line. NK/T, natural killer/T-cell lymphoma/leukemia cell line. C, expression pattern of BC040320 and GPC5 in multiple normal tissues. Expression of BC040320 is hardly visible in normal tissues except for lung, thymus, and lymph node when compared with that of the two cell lines (lanes 1 and 2) with high-level amplification at 13q31-q32.
- Fig. 7.
Exon-intron structure of the C13orf25 gene. A, two expressed sequence tags, BC040320 and AF339828, which are overexpressed in the cell lines with amplification at 13q31.3, are shown above the ····. BC040320 is split into four exons, encompassing two bacterial artificial chromosome clones, RP11–282D2 and RP11–121J7. AF339828 is located to the telomeric side of BC040320 and ∼300-bp apart from BC040320. The primer set used for reverse transcription-PCR is shown below the exons. B, two transcripts obtained by reverse transcription-PCR. One (Transcript-A) is the same as the BC040320 sequence consisting of four exons containing 965-bp nucleotides. The other (Transcript-B) consists of two exons containing 5058-bp nucleotides. Computer analysis showed that 32-AA polypeptides of bA121J7.2 (Vega gene ID) were encoded in the Transcript-A cDNA. Possible open reading frames are shown as
. Five precursor microRNAs (miRNAs; miR91-precursor-13 micro RNA, miR18- precursor-13 micro RNA, miR19a- precursor-13 micro RNA, miR19b- precursor-13 micro RNA, and miR92- precursor-13 micro RNA), including seven mature microRNAs (microRNA miR-17, miR-91, miR-18, miR-19a, miR-20, miR-19b, and miR-92) were obtained from the Transcript-B sequence, and are shown by the ▪ in Transcript-B. C, polypeptides sequences are also shown below the structure. The polypeptides of 13 amino acids (AA) are shared by Transcript-A and Transcript-B, and are indicated by underlining.
Tables
- Table 1
RT-PCRa analysis
BAC clones, EST and STS Marker refer to the information obtained from Ensembl genome data resource. All BAC clones are included in RPCI-11 human male BAC library.
BAC EST/gene STS Marker Forward primer Reverse primer FB Rec1 Karpas 1718 OCI-Ly4 360A9 AW664738 D13S1457 5′-gccacatggtcaggttaaac 5′-cttcactaccttgcgactct − ND ND − 27D9 AA309162 5′-ggctctcatttagctaaatg 5′-aagaagtggttgagacaaca +b + + + 15N8 No EST 321E13 AW236754 D13S1175 5′-gagatggcacagcagttgaa 5′-tagttcaaactctacctgca − ND ND − 447M23 No EST 370B1 LOC121723 5′-aactactggtgaggactgca 5′-caagttccccttctgcagaa − ND ND − AW059867 5′-aaggcttagctattatgctg 5′-acaatgaggaaaatctccca ++c ++ ++ ++ 275J18 BC042969 D13S1818 5′-ctggtcacacatccacaatg 5′-cagtggaatctgagtcctag ++ + + ++ AA628299 5′-cagaaggagtgttaagtctg 5′-caagaagaagctgccagtat ++ ++ ++ ++ 143O10 No EST 309H8 BG183515 D13S1239 5′-ctcatgactgtaatcccagc 5′-gtgatgccgtgaaatgagtc + + + + BG191981 5′-ttcagtgacctcactgactg 5′-gaggattttgcagtcatcgc − ND ND − 114G1 LOC160824 D13S767 5′-aggttttgtcagccacacct 5′-gaactatccgtaccttgtcc − ++ + + 75N6 AA398228 5′-ctgtaccattgtgcccagaa 5′-atgactcagtccttctggct − ND ND − 86C3 No EST D13S265 51A2 No EST 18M3 No EST 388D4 LOC144774 5′-cactgtggcagttatacggt 5′-caatggttttccgcaccagt − ND ND − LOC121727 5′-cctgggaaggatggtttctt 5′-ttacaacacaaggggcacac + + + ++ 409J23 No EST 392A19 LOC121729 5′-ggagccattacttcaagacc 5′-agaccagtacttgtccagct + ++ ++ ++ BG776186 5′-tggacacgtgagtgtgtttc 5′-atgagctcaggcagctctat + ++ − + LOC121728 5′-tgataggagacagacctgac 5′-tccagtcatcctgaggtaga − ND ND − BG186078 5′-tcacacagtgtgactcacag 5′-tgatctcctctctgtagtgc ND − − + AA487882 5′-acccaagggaatattgacac 5′-gaagctagggaagcagtatt ND − − + BM542991 5′-tgcagtaggtggcaatctca 5′-tcgacttggactccatgaga − ND ND − 505P2 BM695971 D13S1234 5′-ccttggagtgcttaaggtag 5′-actagggctctttgtagacc ND − − ++ 158A8 AI027278 5′-gctgtgattgcgaagaagtg 5′-tccatatgtgagtgtggcag ND − − + BG927281 5′-acgccaagctctaatacgac 5′-ctcgggctatggtatatgac − ND ND − BI825411 5′-accctggacaggtatggaat 5′-accatgagcacagtgctcaa − ND ND − AA888411 5′-ctcccattgcagttactatg 5′-gtcgacatgttgttgaggtt + + + + 360H15 AW515966 5′-atgttcaccaggctggtctt 5′-actaactctgtgggcttgca − ND ND − BF818219 5′-tctgcccactaacatctggt 5′-cttgagtagctgagaccaca − ND ND − 114C3 No EST 432D3 LOC144776 D13S1190 5′-cacttccttgaaggggtttc 5′-ctctgactcttgggcacaat ND + + + AI126313 5′-ttgagacacagtcctgctct 5′-gcagggcacaatgttttcag − ND ND − 319L6 AV731092 5′-attggtgaagccacctcaaa 5′-caggctaacatggatctagt ND − − + BG941714 5′-agtgcctacttcctaagacc 5′-tcaggaatcagtgcccaaac ND − − + AI262947 5′-ttgtgttcctggtccaccta 5′-acattgggccggtcacttat ND − − + AI493127 5′-tcaaatcaactgcacctcag 5′-cagttcgagacttcttccat − ND ND − 51B13 BX097335 5′-gaagtaggatggtgacacct 5′-ctcagcaagtcatcctttgc − ND ND − 430K10 AL701000 D13S886 5′-tggcctggagtaattagctg 5′-atttagggggtaggagagca ND − − + LOC160827 5′-ctgtgcactatcacttggga 5′-taggctctaagccgttggtt − ND ND − LOC160826 5′-atggaatcaggttccctcca 5′-ctgttcccttcatctgaatg − ND ND − BQ477330 5′-caaagtgctgggattacagg 5′-gccagctttgctgcacatta − ND ND − BQ477741 5′-caacagaagatcggcccttt 5′-actccctgaagcacagcatt ND + + + AV731847 5′-caggcacttgcttaagggat 5′-aactagcctgcttcagcttc − ND ND − BI481522 5′-atgtgaagagaggtctcagc 5′-agcaaaccacctagaggctt ND + + + BU729287 5′-cttagcctaatctccctaggcgga 5′-tatcaggtaggtggtccagtctca ND ++ ++ ++ BM703078 5′-caggatcttgccctgttagt 5′-tatcgggtggcgaacaagat ND + + + AA719672 5′-tgtccttaggtagacattgt 5′-ttcaacctctgagaaaccca ND ND ND − LOC121734 5′-acttcactgtcaacagcgag 5′-agagaccacatgcttgccat ND ++ ++ ++ BE466687 5′-agttctggaggctaaaagtccagt 5′-gccaaaatcacatggagagactac ND + + + 282D2 BF352993 5′-gagaacagtaatttctttcc 5′-tgcaattattggggtaaagc ND ND ND − BC040320 5′-gtcatacacgtggacctaac 5′-ctgaagtctcaagtgggcat ND ++ ++ ++ 121J7 AF339828 5′-ctgacaagttctcagatcac 5′-actctgcatgagcctagatt ND ++ ++ ++ AA701926 5′-agaccctgatggtctcttta 5′-ggctcaatgttttcctacgg ND ++ ++ ++ BF908089 5′-tggaagaaaggacatgaggt 5′-tctcatgaatccatgcccaa ND + + + AW868481 5′-aagtaaatgtgagaagtagc 5′-tgctcatcctcattgtata ND − − + BX107378 5′-taacctgagcagaatccagccttg 5′-atggacccaaatgctgagaggaac ND ++ ++ ++ AA599001 5′-aagagggacttgctgtgttg 5′-tgcacagacggtacagaagt ND ++ ++ + GPC5 5′-cactggcgggtaaaggggac 5′-agtattcagggaactgtcagtcacacc ND + + + AL043638 5′-ccagtctatcattgatggac 5′-gaagtgcctctgtaattgga ND ++ ++ ++ AL708734 5′-gtaatcccagcactttggga 5′-tcttgttcttgtcccccagt ND + + + 487A2 AF339802 D13S1490 5′-tacctgggtaaccaagactc 5′-ctctgttcactgcattgaag ND ++ ++ ++ H56919 5′-tgttgaccgactgagtgaac 5′-ttatggtgaagtccttcccc ND + + ++ AA705439 5′-cgtactctagagttaaccaa 5′-atgattgtaagttccctgag ND ++ ++ ++ W86832 5′-atcctcatttctcaggggct 5′-cctgtctgctctatgaagct ND − − − N49442 5′-tggctgggcagaaatctgaa 5′-tacaggtctgttcgccacat ND ++ ++ ++ N33596 5′-tccctagcaatgtgatgtac 5′-ctaaggtattcctaggctca ND − − + T84913 5′-gtagtaggtagaactgtcct 5′-atctaccctcggcaattttc ND ++ ++ ++ BU656134 5′-tgctagggctggagtacaat 5′-cattttctcttggctcaccc ND ++ ++ ++ 93M14 AW105449 5′-ccagcaactgtaatacatgc 5′-tcttcaaatccttgcctctg ND − + ++ AV754681 5′-acagccttctttggagagtg 5′-tccaagggcacagtggaatt ND − ++ ++ -
a RT-PCR, reverse transcription-PCR; BAC, bacterial artificial chromosome; EST, expressed sequence tag; FB, fetal brain; ND, not done; STS, sequenced tagged site.
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b Detection of a thin band from the result of electrophoresis.
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c Detection of a thick band from the result of electrophoresis.
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- Table 2
Northern blot analysis
Each signal of those ESTsa and GPC-5 was visually evaluated after 1-week expose.
BAC EST/gene Probe size (bp) Size (kb) Northern Blot Placenta Rec1 Karpas 1718 OCI-Ly4 Jurkat ATN-1 RP11-27D9 AA309162 130 − − − − − − − RP11-370B1 AW059867 160 − − − − − − − RP11-275J18 BC042969 440 − − − − − − − AA628299 220 − − − − − − − RP11-309H8 BG183515 440 − − − − − − − RP11-114G1 LOC160824 550 6.5 +++ +++++ ++++ +++ +++ +++ RP11-388D4 LOC121727 420 − − − − − − − RP11-392A19 LOC121729 350 1.5 + − − − − − RP11-158A8 AA888411 300 − − − − − − − RP11-432D3 LOC144776 500 − − − − − − − RP11-430K10 BQ477741 240 − − − − − − − BI481522 210 5.5 + ++ ++ + ++ + BU729287 460 + − − − − − BM703078 470 − − − − − − − LOC121734 240 0.8 + +++ ++ ++ ++ +++ BE466687 390 − − − − − − − RP11-282D2 BC040320 400 6 − +++++ +++++ +++++ − − RP11-121J7 AF339828 410 6 − +++++ +++++ +++++ +/− − AA701926 240 − − − − − − − BF908089 100 − − − − − − − BX107378 420 − − − − − − − AA599001 200 − − − − − − − GPC5 600 5 + + + + + + AL043638 200 − − − − − − AL708734 250 − − − − − − − RP11-487A2 AF339802 320 6 +/− ++ ++ ++ + + H56919 160 − − − − − − − AA705439 290 15 + ++ + +++ +/− +/− N49442 320 15 + ++ + +++ +/− +/− T84913 200 − − − − − − − BU656134 390 − − − − − − − -
a EST, expression sequence tag; BAC, bacterial artificial chromosome.
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Volume 64, Issue 9
