| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Technical Advances |
From the First Department of Surgery*
and the
Third Department of Internal Medicine,
Hokkaido University School of Medicine, Sapporo; and the Divisions of
Cancer-Related Genes
and Gene
Therapy Development,
Institute for Genetic
Medicine, Hokkaido University, Sapporo, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
After Ishioka and colleagues3 first reported an efficient screening method using yeast prototrophy (URA3 marker) for uracil, namely yeast-based stop codon assay, a modification using yeast color selection (ADE2 marker) has been reported for detection of chain-terminating mutations in some specific genes.4-6 Taking advantage of gap repair and production of GOI::ADE2 (EC 4.1.1.21) chimera protein as a visible reporter in yeast, this method permits a quantitative, efficient detection of chain-terminating mutations as red colonies because of accumulation of an ADE2 substrate in vivo in yeast.7 This method requires, however, construction of an expression vector that is specific to each tested GOI (cloning of GOI into the vector), and thus is not immediately applicable to a newly interested gene. To remove this drawback without compromising the reliability of assay, we made two modifications: 1) amplification of GOI by a nested polymerase chain reaction (PCR) procedure with addition of short terminal sequences for gap repair, and 2) utilization of a universal vector for the acceptor of the amplified GOI. We determined optimal conditions of the assay in regard to the specificity and efficiency of gap repair, and confirmed quantitativeness of the mutation detection and its general applicability to a variety of GOI, minimizing the assay background (false positivity). We here present the establishment of the assay named "universal stop codon assay." Taking the prevalence of chain-terminating mutations in the whole mutation spectrum8 into account, we suggest that the assay is able to cover the majority of human genetic diseases including cancers.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This assay utilizes a YCp type yeast expression vector, which is
held in a single copy in a yeast cell and expresses a chimera protein
of the reading frame of a tested gene fragment and ADE2 gene
(phosphoribosylaminoimidazole carboxylase, E.C. 4.1.1.21). To permit an
automatic integration of the gene fragment in-frame to ADE2
gene by homologous recombination in yeast, short sequences homologous
to the vector cut-ends are added to the fragment during PCR
amplification. Failure in producing a complete chimera protein because
of the presence of a chain-terminating mutation in the tested fragment
leads to accumulation of phosphoribosylaminoimidazole in yeast cells,
resulting in red color of a yeast colony (Figure 1, A and B)
.
|
The S. cerevisiae/Escherichia coli shuttle
expression vector pLS381, an immediate origin of pCA57,4
which has URA3 marker, CEN/ARS sequence, Amp-resistant gene, ColE1 Ori,
and ADE2 driven by CYC promoter, was used for construction of vectors
as follows. pLS381 was linearized at the BamHI site between
CYC promoter and ADE2, and a double-strand oligonucleotide
(5'-CACTAAATT AATAATGACC GGCGCC ATGGATTCTA
GAACAGTTG-3') was integrated into the vector in yeast to replace a
SfoI site with a BamHI site. The resultant new
vector, pMT18 (Figure 1a)
, was linearized with SfoI and
dephosphorylated with calf intestinal alkaline phosphatase (Gibco-BRL,
Tokyo, Japan). Complete digestion of pLS381 and pMT18 was confirmed by
an additional agarose gel electrophoresis after gel purification and
calf intestinal alkaline-phosphatase treatment. Yeast strain yPH857 was
transformed with the linearized 50-ng pMT18 or pLS381 in a manner
described below for comparison of self-ligation of the vectors.
To insert a spacer sequence, a double-strand oligonucleotide, 5'-tta ata atg acc ggc GCC... GGA TCC gcc atg gat tct aga-3', flanking a tag sequence (FLAG, 5'-ATG GAT TAC AAG GAT GAC GAC GAT AAG ATC-3') was integrated into the SfoI-linearized pMT18, yielding pMT18flag. This was also linearized with SfoI, treated with calf intestinal alkaline phosphatase, and tested for complete digestion.
Primer Design for Nested PCR
For nested PCR to amplify GOI with addition of sequences for gap
repair, two primer sets (external and internal) were designed by using
a computer program OLIGO ver. 4.1 (MedProbe, Oslo, Norway). External
primers were made in both outer arms encompassing the target site,
giving a special priority to the specificity and the absence of a
secondary structure. Gene-specific parts of internal primers were
designed as their 5'-termini matching the codon frame of a target
sequence (Table 1)
. Length and position
of the primers were selected not to form a dimer of more than 4 bases
in the 3'-termini. To the 5' end of the gene-specific part of an
internal primer, a sequence for gap repair (homologous recombination)
matching the corresponding cut-end of the vector was added as follows.
For gap repair into pMT18, 5'-CAC ACT AAA TTA ATA ATG ACC GGC ATG-3'
(last ATG for translational start) and 5'-ACC AAC TGT TCT AGA ATC CAT
GGC-3' were added to the 5'-termini of forward and reverse primers
respectively. For gap repair into pMT18flag, 5'-GTC GTC ATC CTT GTA ATC
CAT GGC-3' was added to the 5' end of reverse primers.
|
Nucleic Acid Extraction and cDNA Synthesis
Seven breast cancer cell lines (MCF-7, T-47-D, MDA-MB-435s, MDA-MB-231, ZR-75–1, MDA-MB-436, BT549, SKBr-3) and seven colon cancer cell lines (HCT116, HT29, HCT15/DLD1, HCC2998, KM12, SW480, COLO201) were used in this study. Cells were cultured in 10-cm dishes, and genomic DNA and total RNA were extracted with the use of DNAzol and Trizol reagents (Gibco-BRL) according to the manufacturer’s instructions. Three colon cancer tissues and 10 cervical cancer tissues resected by the standard surgical procedure at the First Department of Surgery, Hokkaido University Hospital and the Gynecology Department, University of Indonesia, were snap-frozen in liquid nitrogen and served for nucleic acid extraction. For RNA, cDNA synthesis was done at 37°C for 60 minutes in a 20-µl reaction mixture containing 1 µg RNA, 1x reverse transcriptase (RT) buffer, 7.5 mmol/L dithiothreitol, 2.0 mmol/L MgCl2, 0.5 mmol/L each dNTP, 10 ng/µl random pdN6 primer, and 10 U/µl Moloney murine leukemia virus-reverse transcriptase (Gibco-BRL).
Nested PCR
Amplification of the target gene with addition of sequences for
gap repair was done in a nested PCR procedure using Pfu
TURBO polymerase (Stratagene, La Jolla, CA) on a Thermal Cycler
2400 (Perkin Elmer, Chiba, Japan). The first PCR was done on 100 ng of
genomic DNA or 2 µl of RT product (cDNA) in a 25-µl reaction
mixture containing 1x cloned Pfu buffer, 0.05 U/µl Pfu
TURBO polymerase, 0.2 mmol/L each dNTP, and 0.4 µmol/L each
external primer. PCR cycles consisted of a 40-second initial
denaturation at 95°C (hot start), and then 10 cycles of denaturation
at 95°C for 40 seconds, annealing at an indicated temperature (Table 1)
for 40 seconds, and extension at 78°C for an extension time (2.0
minutes/kb target size); and after-extension at 78°C for 7 minutes.
The second PCR was done with 2 µl of the 100-fold diluted first PCR
product and an internal primer pair, in 35 cycles at the annealing
temperature indicated in Table 1
. Satisfactory amplification was
verified by electrophoresis in a 1% agarose gel and visualization by
ethidium bromide staining under UV light.
Transformation of Yeast
The yeast strain yPH857 [MATa ura3–53 lys2–801
ade2–101 his3-
200 trp1-63 leu2-1 cyh2R] was transformed
with crude PCR product (1 to 5 µl) and the linearized vector (50 ng)
by a lithium acetate/heat-shock method as described
previously.4
Yeast were then plated and grown at 30°C
for 2 to 3 days on a synthetic medium CAu10 (SD; ura-; 0.67% yeast
nitrogen base, 2% glucose, 1% casamino acids, 20 mg/L
L-tryptophane, 10 mg/L adenine, and 2.5% agar).
After color intensification by incubating the culture plate at 4°C,
formed white and red colonies were counted. Assay results were
expressed as percentages of red colonies in more than 200 colonies per
plate.
Recovery of Plasmids and Sequencing
Yeast were digested for 1 hour with zymolyase 100T (Seikagaku-Kogyo, Tokyo, Japan) and plasmids were then recovered by an alkaline lysis procedure (QIAprep plasmid kit; Qiagen GmbH, Hilden, Germany) and introduced into XL-1blue E. coli cells by electroporation. The presence of a gap-repaired insert of the expected size was confirmed by enzyme digestion with SphI and EcoRV. Plasmids were sequenced with a DyeDeoxy terminator kit (Perkin-Elmer) on an ABI 377 automated sequencer (Perkin Elmer/Applied Biosystems). Primers used for sequencing were VF-1, 5'-CTTCTATAGACACGCAAACAC-3' and VR-1, 5'-CAATCATACGTCCCAATTGTC-3, which anneal the vector outside the gap, and primers specific to each gene.
Comparison with the Conventional Stop Codon Assay
The assay was performed for APC gene (codons 1113 to 1493) on genomic DNA extracted from three colon cancer cell lines (COLO201, DLD1, SW480) and three colon cancer tissues (I, H, M). The assay results were compared to those of the yeast color assay specific to APC gene (Furuuchi and colleagues5 ).
Assay for hMSH6
A part of hMSH6 cDNA (codons 170 to 402) was amplified
with nested PCR from RT products of five colon cancer cell lines
(HCT15/DLD1, HCT116, HT29, HCC2998, and KM12) using primers shown in
Table 1
. yPH857 was transformed with each crude PCR product and
linearized assay vector and cultured as described above.
Assay for E-Cadherin
The entire coding region of E-cadherin cDNA consists of 2649 bp (882 amino acids). We divided the region into three parts to amplify 804-, 732-, and 1209-bp fragments for respective codons 8 to 275, 231 to 474, and 464 to 866 in nested PCR procedures. The assay of each part was performed in 10 cases of cervical cancer. For the first part assay, adenine concentration was increased from 10 to 12.5 or 15 µg/ml enough to supplement the partial ADE2 activity observed as pink colonies.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Yeast strain yPH857 was transformed with 50 ng of BamHI-linearized pLS381 (original vector) or SfoI-linearized pMT18 (new vector) in duplicate independent procedures, and resultant yeast colonies were counted for assessment of self-circularization of the vectors with cohesive versus smooth ends in yeast. BamHI-linearized pLS381 yielded 77 and 72 colonies, whereas SfoI-linearized pMT18 gave 1 and 0 colony only.
Fidelity of Gap-Repair
Nested PCR
In the initial stage of the present study, we observed incorporation of a primer dimer or PCR products with artificial mutations into the vector. To solve the problem, we introduced the following modifications into PCR amplification of gene fragments: 1) to avoid primer duplexing, which may result in formation of a short fragment flanked by recombinable sequences at the both ends, we designed primers to anneal each other with no more than 3 bases; and 2) to minimize base-misincorporation, we changed polymerase from Taq to Pfu TURBO, which has a proofreading function. We also reduced the amplicon size to below 2.5 kb.
In some cases of PCR amplification, we had difficulty in obtaining a good amplification of GOI fragment, probably because of the addition of a long gap repair part to the primers. And in such cases, we often observed incorporation of nonspecific PCR product into the vector. We therefore used nested PCR, to maximize the efficiency and specificity of PCR amplification.
Applying these strategies, we amplified middle 1/3 (1.0 kb), upper 2/3
(2.2 kb), and lower 2/3 (2.2 kb) parts of BRCA1 exon 11 with the primer
sets shown in Table 1
, and tested the assay. Consequently, we observed
reduction in percentages of red colonies respectively to 4.0 to 10.7%
(n = 10; mean, 8.1%), 3.8 to 7.3%
(n = 6; mean, 5.5%), and 10.3 to 16.0%
(n = 6; mean, 13.3%). Sequence analysis of the
plasmids recovered from white colonies demonstrated that all of the
plasmids were incorporated with right gene fragments.
Effects of Primer Length on Assay Background
Further to see whether length of sequences used in homologous recombination could affect the fidelity of recombination, we amplified middle 1/3 of BRCA1 exon 11 using a primer set (gBREX11F2 and gBREX11R2) with additional 18-, 24-, 30-, or 39-base recombination sequences at the 5'-termini, from DNA of cell lines MCF-7 and ZR-75–1, and tested the products in the assay. For each cell line, percentages of the background red colonies were respectively 4.8%, 7.9% (18 bases); 9.8%, 7.2% (24 bases); 11.1%, 12.7% (30 bases); and 7.7%, 8.5% (39 bases).
Effects of Primer Length on Fidelity of Homologous Recombination
We then examined the presence of the insert in the recovered plasmids by SphI and EcoRV digestion. Expected sized inserts were confirmed in 20 of 20 white colonies for the 18-base recombination sequences (95% confidence interval 0.832 to 1.000), 40/40 for 24 bases (0.911 to 1.000), 20/20 for 30 bases (0.832 to 1.000), and 18/20 for 39 bases (0.683 to 0.988). Sequence of the plasmids with inserts verified precise recombination of the insert to the expected place without frameshift or mutation. From these data and the consistent data given by Hua and colleagues9 on the efficiency of homologous recombination in yeast, we determined to add 24-base sequences to the gene-specific part of internal primers for homologous recombination in our system thereafter.
Detection of Mutations
Assay for BRCA1 Exon 11 in Breast Cancer Cell Lines
Assays for BRCA1 exon 11 in two parts were performed in
seven breast cancer cell lines. Obtained percentages of red colonies
for upper 2/3 (codons 224 to 967) and lower 2/3 parts (codons 629 to
1366) were, respectively, as follows: 5.5 and 5.9% (T-47-D); 5.4 and
10.4% (MDA-MB435s); 4.3 and 12.4% (MDA-MB-231); 5.0 and 15.4%
(ZR-75–1); 6.2 and 11.6% (MDA-MB-436); 8.2 and 11.1% (BT549); and
8.2 and 18.1% (SKBr-3). Sequence analysis of the plasmids recovered
from red colonies denied the presence of clonal mutation. These results
showed that mutation was absent in this region of BRCA1 gene
in all of the samples tested, and that the false-positive levels
(background red colonies) were <20% for 2.2-kb-long gene fragments
(Figure 2a)
.
|
Assay for APC exon 15 (codons 1113 to 1493) was
performed on three colon cancer cell lines and three colon cancer
tissues, and results were compared with the corresponding results
previously determined by APC color assay.5
Results of the
universal stop codon assay expressed in percentage of red colonies
were: 8.9% for COLO201 (wild-type), 99.1% for HCT15/DLD1 (codon 1414
GGC>GG), 99.5% for SW480 (codon 1338 CAG>TAG), 44.6% for tumor
sample I (codon 1450 CGA>TGA), 42.3% for tumor sample H (codon 1465
AGT>T, Figure 2b
), and 44.6% for tumor sample M (codons 1309 to 1311
GAAAAGA>GA). These results were consistent with the results by
Furuuchi and colleagues5
(respectively, 4.9%, 97.7%,
99.3%, 56.3%, 33.7%, and 32.7%).
Detection of hMSH6 Mutation
Assay for hMSH6 (codons 170 to 402) was performed on
five colon cancer cell lines. RNA was extracted from these cells, and
subjected to RT-PCR amplification of cDNA using the external and
internal primer sets shown in Table 1
. The assay gave red colonies of
55.9% for HCT15, 8.9% for HCT116, 10.1% for HT29, 7.6% for HCC2998,
and 9.8% for KM12. Sequence analysis of the recovered plasmids from
red colonies for HCT15 revealed one base deletion of codon 289/299
(GGCCTG
GGCTG; Figure 2c
).
Detection of E-Cadherin Mutation
Assay for E-cadherin gene was done by dividing the entire coding
region of 2649 bp into overlapping three parts (first part, codons 8 to
275; second part, codons 231 to 474; third part, codons 464 to 866) as
shown in Table 1
. A total of 10 tumors of uterine cervix were subjected
to the study. On testing the first part, wild-type cDNA gave pink
colonies, making discrimination from red colonies difficult. Computer
analysis of the protein secondary structure revealed a massive cluster
of ß-sheet structures in this region (data not shown), suggesting a
stiff structure of the protein that might have interfered with ADE2
enzymatic activity by concealing the catalytic site. This problem was
overcome by using a medium with an increased amount of adenine (12.5 or
15 µg/ml) and a molecular spacer (FLAG sequence) between E-cadherin
cDNA and ADE2 cDNA (pMT18flag). Of 10 cases of cervical
cancer, two cases showed red colonies of >20% in the third part:
52.0% in case 2 and 65.8% in case 6 (Figure 2d)
. Sequence analysis of
the plasmids recovered from red colonies demonstrated one base deletion
(GATTTTGATTT) at codon 552/553 in case 2 and 146-bp deletion (whole
exon 11 skipping) in case 6.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Because the present assay, namely "universal stop codon assay," reports prevalence of mutant allele in a given sample as percentage of red colonies, false positivity (background red colonies) must be suppressed to a minimum. Considering the clonality of mutant-harboring tumor cells and contamination of normal cells in case of somatic mutation, and unbalanced expression of mutant allele because of nonsense-mediated RNA decay4,17 for both somatic and germline mutation, the background is required to be <20%. As homologous recombination into vector utilizes common sequences, the flanked part of amplified PCR product must be precise and specific to the tested gene, devoid of nonspecific amplification of untoward gene fragments with sequences homologous to the primer sequences. To achieve this, we used a nested PCR procedure. In addition, we designed primers free of duplex formation to avoid a primer dimer, which may be incorporated into the assay vector, as seen in the beginning of the study. We also used a proofreading enzyme, Pfu polymerase, to minimize base misincorporation.18 With these means, we successfully achieved low assay background levels in all of the instances of application.
The universal stop codon assay successfully detected mutations in APC, hMSH6, and E-cadherin genes. Quantitativeness of the assay was also satisfactory. As the assay takes only several days beginning with primer design and can process a large number of samples at once, it can serve as a high-throughput screening method of mutations in human genetic diseases. Although the spectrum of mutations ranges from cytogenetically visible chromosomal arrangements to single-base alterations, the most prevalent mutation type in human genetic diseases is short-base deletion/insertion or base substitution leading to protein-truncation, accounting for >50%.8 As this assay itself provides an efficient cloning system of gene fragments spanning up to 2 kb, it facilitates sequence determination of missense mutations from yeast white colonies as well as nonsense mutations from red colonies.
In conclusion, the universal stop codon assay provides a powerful means of general utility for diagnosis of genetic diseases caused by germline mutation or somatic mutation of genes. This might bring to a solution, at least in part, for increasing demand of genetic diagnosis in the postgenomic era.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Yeast strain and vectors used in this study are available by request for research purposes.
Accepted for publication July 10, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  V.-K. Ton and R. Rao Functional expression of heterologous proteins in yeast: insights into Ca2+ signaling and Ca2+-transporting ATPases Am J Physiol Cell Physiol, September 1, 2004; 287(3): C580 - C589. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Yamada, K. Furuuchi, T. Aoyama, A. Kataoka, J.-i. Hamada, M. Tada, S. Okushiba, S. Kondo, T. Moriuchi, and H. Katoh Reconstructed {beta}-Catenin/TCF4 Signaling in Yeast Applicable to Functional Evaluation of APC Mutations Am. J. Pathol., December 1, 2003; 163(6): 2201 - 2209. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
