The structure of the SBP-Tag-streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits.

The 38-residue SBP-Tag binds to streptavidin more tightly (K(d) -/= 2.5-4.9 nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75 Å resolution shows that the SBP-Tag binds to streptavidin in an unprecedented manner by simultaneously interacting with biotin-binding pockets from two separate subunits. An N-terminal HVV peptide sequence (residues 12-14) and a C-terminal HPQ sequence (residues 31-33) form the bulk of the direct interactions between the SBP-Tag and the two biotin-binding pockets. Surprisingly, most of the peptide spanning these two sites (residues 17-28) adopts a regular α-helical structure that projects three leucine side chains into a groove formed at the interface between two streptavidin protomers. The crystal structure shows that residues 1-10 and 35-38 of the original SBP-Tag identified through in vitro selection and deletion analysis do not appear to contact streptavidin and thus may not be important for binding. A 25-residue peptide comprising residues 11-34 (SBP-Tag2) was synthesized and shown using surface plasmon resonance to bind streptavidin with very similar affinity and kinetics when compared with the SBP-Tag. The SBP-Tag2 was also added to the C-terminus of ß-lactamase and was shown to be just as effective as the full-length SBP-Tag in affinity purification. These results validate the molecular structure of the SBP-Tag-streptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.

Genomic instability in laminopathy-based premature aging.

Premature aging syndromes often result from mutations in nuclear proteins involved in the maintenance of genomic integrity. Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Lack of functional Zmpste24, a metalloproteinase responsible for the maturation of prelamin A, also results in progeroid phenotypes in mice and humans. We found that Zmpste24-deficient mouse embryonic fibroblasts (MEFs) show increased DNA damage and chromosome aberrations and are more sensitive to DNA-damaging agents. Bone marrow cells isolated from Zmpste24-/- mice show increased aneuploidy and the mice are more sensitive to DNA-damaging agents. Recruitment of p53 binding protein 1 (53BP1) and Rad51 to sites of DNA lesion is impaired in Zmpste24-/- MEFs and in HGPS fibroblasts, resulting in delayed checkpoint response and defective DNA repair. Wild-type MEFs ectopically expressing unprocessible prelamin A show similar defects in checkpoint response and DNA repair. Our results indicate that unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging.

Characterization of rearrangements involving 4q, 13q and 16q in hepatocellular carcinoma cell lines using region-specific multiplex-FISH probes.

Deletions in 4q, 13q and 16q were frequently detected in hepatocellular carcinoma (HCC) by comparative genomic hybridization (CGH) studies. However, detailed chromosome structural aberrations are not fully explored. Using CGH combined with multiplex-color FISH (M-FISH) with chromosome region-specific probes (CRPs), chromosome structural aberrations in 4q, 13q and 16q in six HCC cell lines were studied. All CRPs, which were generated from microdissected DNA, were specific, strong in intensity and sensitive enough to detect chromosome structural aberrations including translocation and deletion. FISH with BAC probes was used to further characterize translocation breakpoints and deletions. A breakpoint at 16q22 was localized at a BAC clone (RP11-341K23) and another breakpoint at 4q28 was localized within a 620 kb-region. A minimal deleted region at 13q21 was found between BAC clones RP11-240M20 and RP11-435P18. This study demonstrated that the combination of CGH, M-FISH and BAC-FISH is a very useful tool to detect and characterize translocation breakpoint.

Characterization of 3p, 5p, and 3q in two nasopharyngeal carcinoma cell lines, using region-specific multiplex fluorescence in situ hybridization probes.

Amplification of chromosome arms 3q and 5p and deletion of 3p were frequently detected in nasopharyngeal carcinoma (NPC) with comparative genomic hybridization and loss of heterozygosity studies. To identify the minimal amplified or deleted regions in these arms, structural aberrations in chromosome arms 3p, 3q, and 5p in two NPC cell lines, CNE1 and SUNE1, were studied with multiplex-color FISH (M-FISH) and chromosome region-specific probes (CRP). All CRPs, which were generated from microdissected DNA, were specific and strong in intensity, and sensitive enough to detect chromosome aberrations including translocations, deletions, and amplifications of target regions. In these two NPC cell lines, minimal regions of deletion and amplification were found at 3p12 and 3q26 approximately q27, respectively. On 5p, most of the regions were amplified as intact copies. Interregion translocations of these three arms were also observed. The amplification on 3q26 approximately q27 provided useful hints for further screening the minimal amplification at RP11-115J24 (3q26.2), containing candidate oncogene eIF-5A2. M-FISH with CRPs is thus not only useful in revealing a comprehensive picture of structural aberrations in target chromosomes, but also in narrowing down the minimal region for screening cancer-related genes.

[Molecular cytogenetic analysis for a familial complex chromosomal rearrangement].

OBJECTIVE: To determine a complex chromosomal rearrangement by advanced molecular cytogenetic techniques and analyze its clinical effect. METHODS: A complex chromosomal rearrangement (CCR) involved in chromosomes 5, 16 and 20 in a 29-year-old male carrier was determined by chromosomal microdissection and multicolor fluorescence in situ hybridization (M-FISH), and family degree investigation was further performed. RESULTS: The karyotype of the case was a complex chromosomal translocation among chromosomes 5, 20 and 16, and accompanied with a band of chromosome 20 inserted into chromosome 5. His mother and sister both had the same abnormal karyotype by familial investigation. CONCLUSION: The combined use of M-FISH and chromosome microdissection is a powerful tool to determine CCR. The complex chromosomal rearrangement could be transmitted stably in the family, but still the carriers could give birth to a healthy baby by chance.

Establishment of cell lines from a primary hepatocellular carcinoma and its metastatis.

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world with a very poor prognosis that has been associated with tumor metastasis. The molecular mechanism of HCC metastasis is still unclear. In this study, we established cell lines from a primary tumor (H2-P) and its metastasis (H2-M). G-banding karyotyping, comparative genomic hybridization, and fluorescence in situ hybridization were applied to study these two cell lines and the results demonstrated that they are of the same origin. These cell lines provide a very useful tool to identify genetic alterations associated with HCC metastasis.

Development of a LytE-based high-density surface display system in Bacillus subtilis.

The three N-terminal, tandemly arranged LysM motifs from a Bacillus subtilis cell wall hydrolase, LytE, formed a cell wall-binding module. This module, designated CWBM(LytE), was demonstrated to have tight cell wall-binding capability and could recognize two classes of cell wall binding sites with fivefold difference in affinity. The lower-affinity sites were approximately three times more abundant. Fusion proteins with ß-lactamase attached to either the N- or C-terminal end of CWBM(LytE) showed lower cell wall-binding affinity. The number of the wall-bound fusion proteins was less than that of CWBM(LytE). These effects were less dramatic with CWBM(LytE) at the N-terminal end of the fusion. Both CWBM(LytE) and ß-lactamase were essentially functional whether they were at the N- or C-terminal end of the fusion. In the optimal case, 1.2 × 10(7) molecules could be displayed per cell. As cells overproducing CWBM(LytE) and its fusions formed filamentous cells (with an average of nine individual cells per filamentous cell), 1.1 × 10(8)ß-lactamase molecules could be displayed per filamentous cell. Overproduced CWBM(LytE) and its fusions were distributed on the entire cell surface. Surface exposure and accessibility of these proteins were confirmed by immunofluorescence microscopy.

Generation of a complete set of human telomeric band painting probes by chromosome microdissection.

Chromosomal rearrangements involving telomeric bands have been frequently detected in many malignancies and congenital diseases. To develop a useful tool to study chromosomal rearrangements within the telomeric band effectively and accurately, a whole set of telomeric band painting probes (TBP) has been generated by chromosome microdissection. The intensity and specificity of these TBPs have been tested by fluorescence in situ hybridization and all TBPs showed strong and specific signals to target regions. TBPs of 6q and 17p were successfully used to detect the loss of the terminal band of 6q in a hepatocellular carcinoma cell line and a complex translocation involving the 17p terminal band in a melanoma cell line. Meanwhile, the TBP of 21q was used to detect a de novo translocation, t(12;21), and the breakpoint at 21q was located at 21q22.2. Further application of these TBPs should greatly facilitate the cytogenetic analysis of complex chromosome rearrangements involving telomeric bands.