Comparison of Established Cell Lines at Different Passages by Karyotype and Comparative Genomic Hybridization

Two established cancer cell lines, MCF-7 and Ishikawa, were both obtained directly from a cell repository and through another laboratory. The karyotypes from the two MCF-7 cell lines had up to 83 chromosomes and similarities for chromosomal gain and structural abnormalities. The two Ishikawa cell lines had up to 60 chromosomes with only a missing X as the common chromosome abnormality. CGH studies were performed by co-hybridizing the two Ishikawa or MCF-7 cell lines to normal metaphases. The differences seen between the two MCF-7 cell cultures reflect changes due to passage number and culture conditions. For Ishikawa, DNA polymorphic data and mutation studies suggest that the two cell lines are not derived from the same established tumor cell line. Our study shows the utilization of CGH in comparing cell lines originating from the same specimen. Our study also demonstrates the necessity for periodically evaluating cell lines to confirm their origin.     

西瓜与近缘种亲缘关系的比较基因组原位杂交分析

物种间亲缘关系的研究是杂交育种的理论基础,野生西瓜在西瓜育种中具有重要作用,然而目前对西瓜属物种间亲缘关系的研究十分有限,而且对西瓜属物种的分类问题还存在分歧.比较基因组原位杂交是分析物种间亲缘关系的有效手段,本研究以西瓜基因组DNA作探针,分别对缺须西瓜、热迷西瓜、药西瓜和诺丹西瓜有丝分裂中期染色体进行了比较基因组原位杂交分析,揭示了西瓜属物种间的亲缘关系,同时对分类地位尚存在争议的诺丹西瓜的归属问题进行了分析,发现诺丹西瓜和甜瓜之间具有非常近的亲缘关系,本研究结果为西瓜与近缘种间的远缘杂交提供了重要的理论依据.     

寡核苷酸阵列比较基因组杂交技术及其应用

阵列-比较基因组杂交技术（array comparative genomic hybridization, array CGH）能在全基因组水平和/或高分辨率基础上检测染色体拷贝数的变化,主要应用于遗传学和肿瘤学研究。Array CGH中微阵列探针通常是PCR扩增的BAC克隆或cDNA分子。最近几年,寡核苷酸阵列比较基因组杂交（oligonucleotide array CGH, oaCGH）逐渐开始应用。oaCGH与BAC array CGH比较,具有操作更简便、探针设计更灵活、分辨率更高等多项优点,预计oaCGH将逐步取代利用BAC克隆片段或cDNA分子的array CGH。oaCGH的应用及其与其它高通量检测技术的结合将促进新的癌症相关基因、肿瘤耐药基因的发现。本文综述了现有主要oaCGH平台在空间分辨率、探针长度、灵敏度、特异性等方面的特点及其应用,概括了oaCGH近年来的进展。     

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome ¹. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases ². The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments ². These BAC targets are individually synthesized and spotted in duplicate on a single glass slide ^2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position ^5,6. The SMRT array can detect alterations as small as 50 kb in size ⁷. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection ^7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection ⁸. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.Download video file.^{(62M, mov)}     

鼻咽癌转移相关基因的比较基因组杂交和cDNA微阵列研究

为筛选在鼻咽癌转移中起重要作用的基因 ,应用比较基因组杂交 (CGH)和cDNA微阵列方法研究成瘤和转移能力不同的鼻咽癌细胞株 6 10B和 5 8F .CGH结果显示 ,2种细胞株在 5p、7q、8p、9q、11p、12q和 17p的一定区域存在差异性扩增 ,在 2q存在差异性缺失 ;cDNA微阵列显示 ,相对于非转移性 6 10B细胞株 ,高转移性 5 8F细胞表现一些基因表达的上调及下调 ,CGH和cDNA微阵列的结合是筛选转移相关基因较好方法     

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient’s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.     

Analysis of Molecular Cytogenetic Alteration in Rhabdomyosarcoma by Array Comparative Genomic Hybridization

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma with poor prognosis. The genetic etiology of RMS remains largely unclear underlying its development and progression. To reveal novel genes more precisely and new therapeutic targets associated with RMS, we used high-resolution array comparative genomic hybridization (aCGH) to explore tumor-associated copy number variations (CNVs) and genes in RMS. We confirmed several important genes by quantitative real-time polymerase chain reaction (QRT-PCR). We then performed bioinformatics-based functional enrichment analysis for genes located in the genomic regions with CNVs. In addition, we identified miRNAs located in the corresponding amplification and deletion regions and performed miRNA functional enrichment analysis. aCGH analyses revealed that all RMS showed specific gains and losses. The amplification regions were 12q13.12, 12q13.3, and 12q13.3–q14.1. The deletion regions were 1p21.1, 2q14.1, 5q13.2, 9p12, and 9q12. The recurrent regions with gains were 12q13.3, 12q13.3–q14.1, 12q14.1, and 17q25.1. The recurrent regions with losses were 9p12–p11.2, 10q11.21–q11.22, 14q32.33, 16p11.2, and 22q11.1. The mean mRNA level of GLI1 in RMS was 6.61-fold higher than that in controls (p = 0.0477) by QRT-PCR. Meanwhile, the mean mRNA level of GEFT in RMS samples was 3.92-fold higher than that in controls (p = 0.0354). Bioinformatic analysis showed that genes were enriched in functions such as immunoglobulin domain, induction of apoptosis, and defensin. Proto-oncogene functions were involved in alveolar RMS. miRNAs that located in the amplified regions in RMS tend to be enriched in oncogenic activity (miR-24 and miR-27a). In conclusion, this study identified a number of CNVs in RMS and functional analyses showed enrichment for genes and miRNAs located in these CNVs regions. These findings may potentially help the identification of novel biomarkers and/or drug targets implicated in diagnosis of and targeted therapy for RMS.     

DNA Copy Number Aberrations in Breast Cancer by Array Comparative Genomic Hybridization

Array comparative genomic hybridization (CGH) has been popularly used for an-alyzing DNA copy number variations in diseases like cancer. In this study, we investigated 82 sporadic samples from 49 breast cancer patients using 1-Mb reso-lution bacterial artificial chromosome CGH arrays. A number of highly frequent genomic aberrations were discovered, which may act as "drivers" of tumor pro-gression. Meanwhile, the genomic profiles of four "normal" breast tissue samples taken at least 2 cm away from the primary tumor sites were also found to have some genomic aberrations that recurred with high frequency in the primary tu-mors, which may have important implications for clinical therapy. Additionally, we performed class comparison and class prediction for various clinicopathological pa-rameters, and a list of characteristic genomic aberrations associated with different clinicopathological phenotypes was compiled. Our study provides clues for further investigations of the underlying mechanisms of breast carcinogenesis.     

Preimplantation Genetic Diagnosis for Aneuploidy and Translocations Using Array Comparative Genomic Hybridization

At least 50% of human embryos are abnormal, and that increases to 80% in women 40 years or older. These abnormalities result in low implantation rates in embryos transferred during in vitro fertilization procedures, from 30% in women <35 years to 6% in women 40 years or older. Thus selecting normal embryos for transfer should improve pregnancy results. The genetic analysis of embryos is called Preimplantation Genetic Diagnosis (PGD) and for chromosome analysis it was first performed using FISH with up to 12 probes analyzed simultaneously on single cells. However, suboptimal utilization of the technique and the complexity of fixing single cells produced conflicting results. PGD has been invigorated by the introduction of microarray testing which allows for the analysis of all 24 chromosome types in one test, without the need of cell fixation, and with staggering redundancy, making the test much more robust and reliable. Recent data published and presented at scientific meetings has been suggestive of increased implantation rates and pregnancy rates following microarray testing, improvements in outcome that have been predicted for quite some time. By using markers that cover most of the genome, not only aneuploidy can be detected in single cells but also translocations. Our validation results indicate that array CGH has a 6Mb resolution in single cells, and thus the majority of translocations can be analyzed since this is also the limit of karyotyping. Even for translocations with smaller exchanged fragments, provided that three out of the four fragments are above 6Mb, the translocation can be detected.     

拟南芥DNA探针的比较基因组原位杂交揭示的拟南芥与远缘植物基因组间的同源性

采用生物素标记的拟南芥基因组DNA探针在75%杂交严谨度下对双子叶植物番茄、蚕豆和单子叶植物水稻、玉米、大麦的染色体进行了比较基因组荧光原位杂交（comparative genomic in situ hybridization,cGISH）分析,以揭示拟南芥与远缘植物基因组间的同源性.cGISH信号代表了拟南芥基因组DNA中的重复DNA与靶物种染色体上同源序列的杂交.探针DNA在所有靶物种的全部染色体上都产生了杂交信号.杂交信号为散在分布,并呈现随基因组增大,杂交信号增多,且分布更加分散的趋势.所有靶物种的核仁组织区（NOR）都显示了明显强于其他区域的杂交信号,表明拟南芥基因组DNA探针可用于植物NOR的物理定位.在所有的靶物种中,信号主要分布在染色体的臂中间区和末端,着丝粒或近着丝粒区有少数信号分布.大麦染色体显示了与C-和N-带不同的独特的cGISH信号带型,表明此探针可用于不同植物染色体的识别.这些结果表明,拟南芥基因组与远缘植物基因组之间,除rDNA和端粒重复序列外,还存在其它同源的重复DNA;一些重复DNA序列在被子植物分歧进化为单子叶和双子叶植物之前就已存在,虽经历了长期的进化过程,至今在远缘物种之间仍保持了较高的同源性.结果还提示,大基因组中古老而保守的重复DNA在进化过程中发生了明显的扩增.     

Mutation Screening and Array Comparative Genomic Hybridization Using a 180K Oligonucleotide Array in VACTERL Association

In order to identify genetic causes of VACTERL association (V vertebral defects, A anorectal malformations, C cardiac defects, T tracheoesofageal fistula, E esophageal atresia, R renal anomalies, L limb deformities), we have collected DNA samples from 20 patients diagnosed with VACTERL or with a VACTERL-like phenotype as well as samples from 19 aborted fetal cases with VACTERL. To investigate the importance of gene dose alterations in the genetic etiology of VACTERL association we have performed a systematic analysis of this cohort using a 180K array comparative genomic hybridization (array-CGH) platform. In addition, to further clarify the significance of PCSK5, HOXD13 and CHD7 genes in the VACTERL phenotype, mutation screening has been performed. We identified pathogenic gene dose imbalances in two fetal cases; a hemizygous deletion of the FANCB gene and a (9;18)(p24;q12) unbalanced translocation. In addition, one pathogenic mutation in CHD7 was detected, while no apparent disease-causing mutations were found in HOXD13 or PCSK5. Our study shows that although large gene dose alterations do not seem to be a common cause in VACTERL association, array-CGH is still important in clinical diagnostics to identify disease cause in individual cases.     

Broad Conservation of Milk Utilization Genes in Bifidobacterium longum subsp. infantis as Revealed by Comparative Genomic Hybridization

Human milk oligosaccharides (HMOs) are the third-largest solid component of milk. Their structural complexity renders them nondigestible to the host but liable to hydrolytic enzymes of the infant colonic microbiota. Bifidobacteria and, frequently, Bifidobacterium longum strains predominate the colonic microbiota of exclusively breast-fed infants. Among the three recognized subspecies of B. longum, B. longum subsp. infantis achieves high levels of cell growth on HMOs and is associated with early colonization of the infant gut. The B. longum subsp. infantis ATCC 15697 genome features five distinct gene clusters with the predicted capacity to bind, cleave, and import milk oligosaccharides. Comparative genomic hybridizations (CGHs) were used to associate genotypic biomarkers among 15 B. longum strains exhibiting various HMO utilization phenotypes and host associations. Multilocus sequence typing provided taxonomic subspecies designations and grouped the strains between B. longum subsp. infantis and B. longum subsp. longum. CGH analysis determined that HMO utilization gene regions are exclusively conserved across all B. longum subsp. infantis strains capable of growth on HMOs and have diverged in B. longum subsp. longum strains that cannot grow on HMOs. These regions contain fucosidases, sialidases, glycosyl hydrolases, ABC transporters, and family 1 solute binding proteins and are likely needed for efficient metabolism of HMOs. Urea metabolism genes and their activity were exclusively conserved in B. longum subsp. infantis. These results imply that the B. longum has at least two distinct subspecies: B. longum subsp. infantis, specialized to utilize milk carbon, and B. longum subsp. longum, specialized for plant-derived carbon metabolism.The newborn infant not only tolerates but requires colonization by commensal microbes for its own development and health (3). The relevance of the gut microbiome in health and disease is reflected by its influence in a number of important physiological processes, from physical maturation of the developing immune system (28) to the altered energy homeostasis associated with obesity (51, 52).Human milk provides all the nutrients needed to satisfy the neonate energy expenditure and a cadre of molecules with nonnutritional but biologically relevant functions (6). Neonatal health is likely dependent on the timely and complex interactions among bioactive components in human milk, the mucosal immune system, and specialized gut microbial communities (30). Human milk contains complex prebiotic oligosaccharides that stimulated the growth of select bifidobacteria (24, 25) and are believed to modulate mucosal immunity and protect the newborn against pathogens (23, 33, 41). These complex oligosaccharides, which are abundantly present in human milk (their structures are reviewed by Ninonuevo et al. [31] and LoCascio et al. [24]), arrive intact in the infant colon (5) and modulate the composition of neonatal gastrointestinal (GI) microbial communities.Bifidobacteria and, frequently, Bifidobacterium longum strains often predominate the colonic microbiota of exclusively breast-fed infants (10, 11). Among the three subspecies of B. longum, only B. longum subsp. infantis grows robustly on human milk oligosaccharides (HMOs) (24, 25). The availability of the complete genome sequences of B. longum subsp. infantis ATCC 15697 (40) and two other B. longum subsp. longum strains (22, 39) made possible the analysis of whole-genome diversity across the B. longum species. Analysis of the B. longum subsp. infantis ATCC 15697 genome has identified regions predicted to enable the metabolism of HMOs (40); however, their distribution across the B. longum spp. remains unknown. We predict that these regions are exclusively conserved in B. longum strains adapted to colonization of the infant gut microbiome and are therefore capable of robust growth on HMOs. In this work, whole-genome microarray comparisons (comparative genomic hybridizations [CGHs]) were used to associate genotypic biomarkers among 15 B. longum strains exhibiting various HMO utilization phenotypes and host associations.     

Microdeletion and Microduplication Analysis of Chinese Conotruncal Defects Patients with Targeted Array Comparative Genomic Hybridization

Objective

The current study aimed to develop a reliable targeted array comparative genomic hybridization (aCGH) to detect microdeletions and microduplications in congenital conotruncal defects (CTDs), especially on 22q11.2 region, and for some other chromosomal aberrations, such as 5p15-5p, 7q11.23 and 4p16.3.

Methods

Twenty-seven patients with CTDs, including 12 pulmonary atresia (PA), 10 double-outlet right ventricle (DORV), 3 transposition of great arteries (TGA), 1 tetralogy of Fallot (TOF) and one ventricular septal defect (VSD), were enrolled in this study and screened for pathogenic copy number variations (CNVs), using Agilent 8 x 15K targeted aCGH. Real-time quantitative polymerase chain reaction (qPCR) was performed to test the molecular results of targeted aCGH.

Results

Four of 27 patients (14.8%) had 22q11.2 CNVs, 1 microdeletion and 3 microduplications. qPCR test confirmed the microdeletion and microduplication detected by the targeted aCGH.

Conclusion

Chromosomal abnormalities were a well-known cause of multiple congenital anomalies (MCA). This aCGH using arrays with high-density coverage in the targeted regions can detect genomic imbalances including 22q11.2 and other 10 kinds CNVs effectively and quickly. This approach has the potential to be applied to detect aneuploidy and common microdeletion/microduplication syndromes on a single microarray.     

Genomic Profiling of Oral Squamous Cell Carcinoma by Array-Based Comparative Genomic Hybridization

We designed a study to investigate genetic relationships between primary tumors of oral squamous cell carcinoma (OSCC) and their lymph node metastases, and to identify genomic copy number aberrations (CNAs) related to lymph node metastasis. For this purpose, we collected a total of 42 tumor samples from 25 patients and analyzed their genomic profiles by array-based comparative genomic hybridization. We then compared the genetic profiles of metastatic primary tumors (MPTs) with their paired lymph node metastases (LNMs), and also those of LNMs with non-metastatic primary tumors (NMPTs). Firstly, we found that although there were some distinctive differences in the patterns of genomic profiles between MPTs and their paired LNMs, the paired samples shared similar genomic aberration patterns in each case. Unsupervised hierarchical clustering analysis grouped together 12 of the 15 MPT-LNM pairs. Furthermore, similarity scores between paired samples were significantly higher than those between non-paired samples. These results suggested that MPTs and their paired LNMs are composed predominantly of genetically clonal tumor cells, while minor populations with different CNAs may also exist in metastatic OSCCs. Secondly, to identify CNAs related to lymph node metastasis, we compared CNAs between grouped samples of MPTs and LNMs, but were unable to find any CNAs that were more common in LNMs. Finally, we hypothesized that subpopulations carrying metastasis-related CNAs might be present in both the MPT and LNM. Accordingly, we compared CNAs between NMPTs and LNMs, and found that gains of 7p, 8q and 17q were more common in the latter than in the former, suggesting that these CNAs may be involved in lymph node metastasis of OSCC. In conclusion, our data suggest that in OSCCs showing metastasis, the primary and metastatic tumors share similar genomic profiles, and that cells in the primary tumor may tend to metastasize after acquiring metastasis-associated CNAs.     

Increasing Live Birth Rate by Preimplantation Genetic Screening of Pooled Polar Bodies Using Array Comparative Genomic Hybridization

Meiotic errors during oocyte maturation are considered the major contributors to embryonic aneuploidy and failures in human IVF treatment. Various technologies have been developed to screen polar bodies, blastomeres and trophectoderm cells for chromosomal aberrations. Array-CGH analysis using bacterial artificial chromosome (BAC) arrays is widely applied for preimplantation genetic diagnosis (PGD) using single cells. Recently, an increase in the pregnancy rate has been demonstrated using array-CGH to evaluate trophectoderm cells. However, in some countries, the analysis of embryonic cells is restricted by law. Therefore, we used BAC array-CGH to assess the impact of polar body analysis on the live birth rate. A disadvantage of polar body aneuploidy screening is the necessity of the analysis of both the first and second polar bodies, resulting in increases in costs for the patient and complex data interpretation. Aneuploidy screening results may sometimes be ambiguous if the first and second polar bodies show reciprocal chromosomal aberrations. To overcome this disadvantage, we tested a strategy involving the pooling of DNA from both polar bodies before DNA amplification. We retrospectively studied 351 patients, of whom 111 underwent polar body array-CGH before embryo transfer. In the group receiving pooled polar body array-CGH (aCGH) analysis, 110 embryos were transferred, and 29 babies were born, corresponding to live birth rates of 26.4% per embryo and 35.7% per patient. In contrast, in the control group, the IVF treatment was performed without preimplantation genetic screening (PGS). For this group, 403 embryos were transferred, and 60 babies were born, resulting in live birth rates of 14.9% per embryo and 22.7% per patient. In conclusion, our data show that in the aCGH group, the use of aneuploidy screening resulted in a significantly higher live birth rate compared with the control group, supporting the benefit of PGS for IVF couples in addition to the suitability and effectiveness of our polar body pooling strategy.     

Cross-Species Array Comparative Genomic Hybridization Identifies Novel Oncogenic Events in Zebrafish and Human Embryonal Rhabdomyosarcoma

Human cancer genomes are highly complex, making it challenging to identify specific drivers of cancer growth, progression, and tumor maintenance. To bypass this obstacle, we have applied array comparative genomic hybridization (array CGH) to zebrafish embryonal rhabdomyosaroma (ERMS) and utilized cross-species comparison to rapidly identify genomic copy number aberrations and novel candidate oncogenes in human disease. Zebrafish ERMS contain small, focal regions of low-copy amplification. These same regions were commonly amplified in human disease. For example, 16 of 19 chromosomal gains identified in zebrafish ERMS also exhibited focal, low-copy gains in human disease. Genes found in amplified genomic regions were assessed for functional roles in promoting continued tumor growth in human and zebrafish ERMS – identifying critical genes associated with tumor maintenance. Knockdown studies identified important roles for Cyclin D2 (CCND2), Homeobox Protein C6 (HOXC6) and PlexinA1 (PLXNA1) in human ERMS cell proliferation. PLXNA1 knockdown also enhanced differentiation, reduced migration, and altered anchorage-independent growth. By contrast, chemical inhibition of vascular endothelial growth factor (VEGF) signaling reduced angiogenesis and tumor size in ERMS-bearing zebrafish. Importantly, VEGFA expression correlated with poor clinical outcome in patients with ERMS, implicating inhibitors of the VEGF pathway as a promising therapy for improving patient survival. Our results demonstrate the utility of array CGH and cross-species comparisons to identify candidate oncogenes essential for the pathogenesis of human cancer.     

De Novo Identification of Single Nucleotide Mutations in Caenorhabditis elegans Using Array Comparative Genomic Hybridization

Array comparative genomic hybridization (aCGH) has been used primarily to detect copy-number variants between two genomes. Here we report using aCGH to detect single nucleotide mutations on oligonucleotide microarrays with overlapping 50-mer probes. This technique represents a powerful method for rapidly detecting novel homozygous single nucleotide mutations in any organism with a sequenced reference genome.     

Survey of Genomic Diversity among Enterococcus faecalis Strains by Microarray-Based Comparative Genomic Hybridization

We have compared nine Enterococcus faecalis strains with E. faecalis V583 by comparative genomic hybridization using microarrays (CGH). The strains used in this study (the “test” strains) originated from various environments. CGH is a powerful and promising tool for obtaining novel information on genome diversity in bacteria. By CGH, one obtains clues about which genes are present or divergent in the strains, compared to a reference strain (here, V583). The information obtained by CGH is important from both ecological and systematic points of view. CGH of E. faecalis showed considerable diversity in gene content: Compared to V583, the percentage of divergent genes in the test strains varied from 15% to 23%, and 154 genes were divergent in all strains. The main variation was found in regions corresponding to exogenously acquired or mobile DNA in V583. Antibiotic resistance genes, virulence factors, and integrated plasmid genes dominated among the divergent genes. The strains examined showed various contents of genes corresponding to the pTEF1, pTEF2, and pTEF3 genes in V583. The extensive transport and metabolic capabilities of V583 appeared similar in the test strains; CGH indicated that the ability to transport and metabolize various carbohydrates was similar in the test strains (verified by API 50 CH assays). The contents of genes related to stress tolerance appeared similar in V583 and the nine test strains, supporting the view of E. faecalis as an organism able to resist harsh conditions.     

Identification of Brevibacteriaceae by Multilocus Sequence Typing and Comparative Genomic Hybridization Analyses

Multilocus sequence typing with nine selected genes is shown to be a promising new tool for accurate identifications of Brevibacteriaceae at the species level. A developed microarray also allows intraspecific diversity investigations of Brevibacterium aurantiacum showing that 13% to 15% of the genes of strain ATCC 9174 were absent or divergent in strain BL2 or ATCC 9175.Brevibacteriaceae play a major part in the cheese smear community (6, 11). The classification and typing of cheese-related Brevibacteriaceae have been based mainly on molecular methods such as amplified ribosomal DNA restriction enzyme analysis, pulsed-field gel electrophoresis, and ribotyping (8, 10, 12). Recently, the original Brevibacterium linens group was split into two species on the basis of their physiological and biochemical characteristics, the sugar and polyol composition of their teichoic acids, and their 16S rRNA sequence and DNA-DNA hybridization levels. One species remains B. linens and is represented by type strain ATCC 9172. The other, represented by type strain ATCC 9175, has been renamed Brevibacterium aurantiacum. Regarding this new classification, the taxonomic position of cheese-related isolates has to be revisited and potential relationships between phylogenetic affiliation and the potential occurrence of given metabolic characteristics redefined (7). The unfinished genome sequence of B. aurantiacum ATCC 9174 has recently been released by the Joint Genome Institute (http://genome.jgi-psf.org/draft_microbes/breli/breli.home.html). The development of focused phylogenetic approaches using multiple markers in conjunction with whole-genome screening techniques such as comparative genomic hybridization (CGH) has proven to be useful for the detailed characterization of pathogenic species, including food pathogens (3, 5, 9). However, only a few technological species have been investigated at an intraspecies level (2). Our intention was thus to develop modern tools to facilitate the typing of strains of technological interest, for which Brevibacteriaceae could be used as a case study.