Alu Family Of Sequences Research Articles

Abstract P2-01-20: The non-invasive treatment for sentinel lymph node metastasis by photodynamic therapy using a verteporfin solubilized phospholipid polymer aggregate

Abstract Introduction: Sentinel lymph node biopsy (SLNB) has become a standard procedure for axillary lymph node evaluation in clinically node-negative breast cancer patients. Recent trial suggested that patients with 1or 2 sentinel lymph nodes (SLNs) involvement could be treated with SLNB alone. Although SLNB is much less invasive procedure comparing with axillary lymph node dissection (ALND), it is still associated with complications such as lymph edema, numbness and pain. Photodynamic therapy (PDT) against cancer is a non-invasive optical therapeutic method in which the topical or systemic delivery of photosensitizing drugs is followed by its subsequent activation with broadband red light. In this study, the usefulness of PDT for treating SLN metastasis was evaluated in murine model. Materials and Methods: Verteporfin, a hydrophobic photosensitizer (PS) forms a soluble conjugate in aqueous medium with a water-soluble and amphiphilic PMB polymer as a solubilizer. The PMB forms stable and well-dispersed molecular aggregate when its concentration is over 1.0 mg/mL based on the hydrophobic interactions among polymer chains. The verteporfin can form conjugate (PMB-vertepoffin) with hydrophobic domain in the PMB aggregate. The PMB-verteporfin was injected at dorsum manus of BALB/c nude mice. The concentrations of verteporfin in tissues were determined by measuring the fluorescence emitted at 700 nm (with excitation at 430 nm). To develop a murine SLN metastasis model, 5 x 105 human epidermoid carcinoma A431 cells with stable expression of GFP were injected to the forearm of BALB/c nude mice. Seven days after inoculation of cancer cells, 20 μL of PMB-verteporfin was injected at dorsum manus of BALB/c nude mice and 75 J of light energy was delivered using a 640 nm diode laser for a total treatment time of 1 min. Fifty-three mice were randomly assigned to the combination of PMB-verteporfin injection and light exposure (A), light exposure alone (B), PMB-verteporfin injection alone (C), and no treatment (D) groups. Ten days after PDT, brachial lymph nodes, which were considered as SLNs were harvested and evaluated by stereoscopic fluorescence microscope. And, DNA was extracted from harvested lymph node. Human Alu family sequence was detected by 7300 Real Time PCR system (Applied Biosystems, Carlsbad CA USA) to estimate metastatic volume. Results: The concentration of verteporfin in SLN was siginificantly higher than other organs including lung, liver, kidney and brachial skin. The group A significantly reduced the SLN metastasis (13%) comparing with , group B (57%), group C (46%) and group D (52%). The Ct value in a PCR of the combination of group A (Ct=29.17) significantly reduced the SLN metastasis comparing with group B (Ct=22.45, p=0.018), group C (Ct=25.58, p=0.018) and group D (Ct=25.54, p=0.005). Conclusions: These data suggested that PDT using PMB as a nanotransporter of verteporfin could be a minimally invasive treatment of SLN metastasis in breast cancer, and represent a potential alternative procedure to SLNB. Citation Format: Shimada K, Matsuda S, Jinno H, Konno T, Ito A, Arai T, Ishihara K, Kitagawa Y. The non-invasive treatment for sentinel lymph node metastasis by photodynamic therapy using a verteporfin solubilized phospholipid polymer aggregate [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-01-20.

A short guide to the human genome

A short guide to the human genome

Structure of the human retinoblastoma-related p107 gene and its intragenic deletion in a B-cell lymphoma cell line

The human p107 protein shares many structural and functional features with the retinoblastoma gene product and retinoblastoma-related p130 protein. In this study, we have cloned and elucidated the complete intron–exon organization of the gene encoding the p107 protein. The gene contains 22 exons spanning over 100 kilobase pairs of genomic DNA. The length of individual exons ranges from 50 to 840 base pairs. The arrays of exons in the p107 gene are rather similar among members of the gene family, especially to those of the p130 gene, while the length of introns is extensively diverse. This study will provide a molecular basis for implementing comprehensive screening for p107 mutations using genomic DNAs from human malignancies. We also show a detailed structure of an intragenic deletion of the p107 gene found in a human B-cell lymphoma cell line, KAL-1, which was shown to occur by homologous recombination between the two directly repeated Alu family sequences.

Ku antigen binds to Alu family DNA.

The GC-rich segment containing GGAGGC (Alu core) is conserved within the RNA polymerase III (pol III) promoters of Alu family sequences. We have shown that the GGAGGC motif functions as a modulator of DNA replication as well as of transcription, and identified the proteins binding to the motif in human HeLa cells. In this study, the Alu core binding proteins were partially purified from human Raji cells by using an Alu core DNA affinity column. Both the proteins thus purified were implied to be subunits of Ku antigen based on the following criteria: The molecular weights of the proteins estimated on gel electrophoreses were 70 and 85 kDa, respectively, under denaturing conditions, while under non-denaturing conditions only one band was observed for the same sample at 150 kDa, probably representing hetero-dimer formed between the 70 and 85 kDa proteins. The sizes and the hetero-dimer formation are reminiscent of the 70 and 80 kDa subunits of Ku antigen (Ku-p70 and Ku-p80). Moreover, the purified proteins were immunoreactive with anti-Ku antibodies, and the specific DNA-protein complex on the Alu core element was cancelled by the anti-Ku antibodies. The nucleoprotein complex showed the same clipping patterns as those of the complex between the Alu core element and an authentically purified Ku antigen after proteolytic cleavage with trypsin and chymotrypsin.

A Sequence Analysis of the Genomic Regions Involved in the Rearrangements between TPM3 and NTRK1 Genes Producing TRK Oncogenes in Papillary Thyroid Carcinomas

Papillary thyroid carcinomas have frequently been found to display oncogenic rearrangements of the NTRK1 gene, which encodes the high-affinity nerve growth factor receptor. Replacement of its extracellular domain by sequences coding for the 221 amino-terminal residues of the TPM3 gene was responsible for the oncogenic NTRK1 activation in three of eight of these tumors. In all of them, the illegitimate recombination involved the 611-bp NTRK1 intron placed upstream of the transmembrane domain and the TPM3 intron located between exons 7 and 8. Therefore, due to the splicing mechanism, all of the TPM3/NTRK1 gene fusions encoded an invariable transcript and the same chimeric protein of 70 kDa, which was constitutively phosphorylated on tyrosine. In two of the three tumors the simultaneous presence of the reciprocal products of the TPM3/NTRK1 recombination, 5′TPM3-3′NTRK1 and 5′NTRK1-3′TPM3 sequences, respectively, and the previously demonstrated localization of both genes on the long arm of chromosome 1 lead us to suggest that an intrachromosomal inversion could be responsible for their recombination. In an attempt to understand the molecular basis that predisposes NTRK1 and TPM3 genes to be a recurrent target of illegitimate recombination, we have determined the nucleotide sequence around the breakpoints of the recombination products in all three patients as well as those of the corresponding regions from the normal TPM3 and NTRK1 genes. In these regions, a search for common features usually involved in illegitimate recombination in mammalian cells revealed the presence of some recombinogenic elements as well as palindromes, direct and inverted repeats, and Alu family sequences.

Periodicity of DNA bend sites in human epsilon-globin gene region. Possibility of sequence-directed nucleosome phasing.

Analysis by the circular permutation assay of the human epsilon-globin gene region revealed that the DNA bend sites were located every 682.5 +/- 132.0 base pairs on average, separating the region into domains. Among 10 major and 1 minor bend sites mapped in the region, the transcription initiation and termination sites of the epsilon-globin gene were located close to the bend sites, and the first and the second exons of the epsilon-globin gene were separated from the third exon by another site. The bend sites were also located anterior to the two Alu family sequences. Short poly(dA).poly(dT) tracts typical for DNA bending were not always present in the sites. Fine mapping of a bend site having no poly(dA).poly(dT) tracts with concatenated oligonucleotides and analysis by S1 nuclease nicking assay indicated that the unusual structure, a base slippage or a partial triplex DNA structure, formed by a polypurine.polypyrimidine sequence in the region is the basis of bending. The bend sites were mapped in the promoter region (within approximately 300 base pairs from the cap site) of the human beta-globin and in c-myc and erythropoietin receptor genes, as well as in the mouse beta maj-globin gene. The conservation and the periodicity of the bend sites in the noncoding region suggest the active role of the sites that is a signal for nucleosome phasing.

Generation of random internal deletion derivatives of YACs by homologous targeting to Alu sequences.

To facilitate the manipulation of human genomic DNA in yeast artificial chromosome (YAC) clones, a plasmid to integrate the selective marker for antibiotic G418 resistance into YACs and to delete some of the human DNA fragments from YACs was constructed. The linearized integration/deletion plasmid, which contains Alu family sequences at both ends, can recombine with YACs containing human repetitive sequences via homologous recombination. The homologous recombination results in a random integration of the antibiotic G418-resistant gene into a human genomic Alu sequence, and in most cases, an internal deletion within the YAC. The YACs with internal deletions can be useful to identify the location of the genes if they produce functional knockouts. In those cases when the integration/deletion event disrupts the integrity of the gene so it no longer can produce a viable and functional mRNA in fused eukaryotic cells, the site of integration in the YAC thus serves as a marker for the inactivated gene. In this report we describe a model system to locate specific genes in YACs.

Stimulation of SV40 DNA replication and transcription by Alu family sequence

The sequence motif GGAGGC ( Alu core) is present in the Alu family repeats, where it is required for RNA polymerase III promoter function. This motif is also found in the SV40 origin ( ori) of replication. Here, an oligonucleotide containing the Alu sequence was inserted into pSV2CAT, a plasmid composed of the SV40 enhancer/promoter/ ori linked to the bacterial chloramphenicol acetyltransferase gene (CAT), to see the effect of the Alu sequence on SV40 DNA replication and transcription. Results of transfection experiments in human HeLa cells showed that the Alu sequence stimulated sequence-specifically replication and transcription in the SV40 system. Stimulation effects on DNA replication were observed when the Alu sequence was placed upstream of enhancer/promoter/ ori in either orientation, while effects on transcription were detected only when it was inserted in the normal orientation. These effects correlate with sequence-specific binding of two proteins (40 kDa and 120 kDa) to this motif. In fact, binding was abolished by a mutation in the cognate sequence that disrupted stimulation of replication and transcription. Both proteins bind duplex DNA, while the 40 kDa one also binds the minus strand with high affinity.

Structure of the gene for human complement protein decay accelerating factor.

Decay accelerating factor (DAF) is a glycophospholipid-anchored membrane protein that is part of the regulators of complement activation (RCA) gene family located on human chromosome 1, band q32. These proteins, beginning at their amino terminus, consist largely of multiple copies of an approximately 60 amino acid short consensus repeat (SCR). A DAF cDNA clone was used to identify overlapping bacteriophage genomic clones. The human DAF gene spans approximately 40 kb and consists of 11 exons. The length of these exons and introns varies considerably, with the exons ranging from 21 to 956 bp and the introns ranging from approximately 0.5 to 19.8 kb. SCR I, II, and IV are all encoded by single exons; however, SCR III is encoded by two separate exons, with the splice junction occurring after the second nucleotide of the codon for the glycine residue at position 34 of the consensus sequence. This feature has also been found in CR1, CR2, membrane cofactor protein, and murine factor H. Following the SCR in DAF is a 76 amino acid serine/threonine-rich domain encoded on three separate exons. Exon 10 encodes the Alu family sequence that has been found as an insert in a minor class of DAF cDNA, thus indicating that this mRNA arises by standard alternative splicing. The last DAF exon, which comes after the largest intron of 19.8 kb, encodes the hydrophobic carboxy terminus and the 3'UT region. The nature of the signal that directs posttranslational attachment of a glycophospholipid anchor to DAF is not known, but that signal is apparently spread over three exons and greater than 20 kb. An analysis of the DAF gene provides additional evidence for the common evolutionary heritage of the RCA gene family. The exon/intron structure of this gene will facilitate experiments aimed at understanding the functions of the various domains of DAF.

The organization and the complete nucleotide sequence of the human NADH-cytochrome b5 reductase gene

The organization and the complete nucleotide (nt) sequence of the b5R gene encoding human NADH-cytochrome b5 reductase (b5R; EC 1.6.2.2) have been determined by a combination of restriction mapping and nt sequence analysis of overlapping genomic DNA clones. The entire gene is about 31 kb in length and contains nine exons and eight introns. Exon 2 contains the junction of the membrane-binding domain and the catalytic domain of b5R, indicating that two forms of b5R, a soluble and a membrane-bound form, are generated by post-translational processing. The 5' portion of the b5R gene lacks the canonical 5' transcriptional regulatory elements, but contains five copies of the GC box sequence G-G-G-C-G-G. While the average G + C content of the b5R gene is 55%, that of the 5' portion of the gene is extraordinarily high (86%). The CpG dinucleotide sequence was found at a very high frequency in this G + C-rich region. These structural features are very similar to those of the regulatory regions of constitutively expressed ‘housekeeping’ genes. Several transcription start points were identified by the primer extension experiment. Seventeen complete and twelve incomplete Alu family sequences were found in introns. An uncanonical polyadenylation signal was detected in the 3'-untranslated region of the gene as A-G-T-A-A-A instead of A-A-T-A-A-A.

Induction of an alu-sequence containing transcript by insulin in human hepatoma cells

A cDNA clone, designated AF19-1, was isolated from a cDNA library derived from insulin-stimulated hepatoma cells. The nucleotide sequences of AF19-1 showed 83% homology to Alu consensus sequence. It contained a full-length 300-bp Alu family sequence followed in direct tandem by a partial sequence of Alu left monomer. Primer extension analysis confirmed that this Alu transcript was induced even in short-term insulin stimulation. The increase in Alu-transcripts in the early phase of insulin stimulation in hepatoma cells suggests that the Alu sequences may play some important regulatory roles in gene expression.

Two copies of the human apolipoprotein C-I gene are linked closely to the apolipoprotein E gene.

The gene for human apolipoprotein (apo) C-I was selected from human genomic cosmid and lambda libraries. Restriction endonuclease analysis showed that the gene for apoC-I is located 5.5 kilobases downstream of the gene for apoE. A copy of the apoC-I gene, apoC-I', is located 7.5 kilobases downstream of the apoC-I gene. Both genes contain four exons and three introns; the apoC-I gene is 4653 base pairs long, the apoC-I' gene 4387 base pairs. In each gene, the first intron is located 20 nucleotides upstream from the translation start signal; the second intron, within the codon of Gly-7 of the signal peptide region; and the third intron, within the codon for Arg39 of the mature plasma protein coding region. The upstream apoC-I gene encodes the known apoC-I plasma protein and differs from the downstream apoC-I' gene in about 9% of the exon nucleotide positions. The most important difference between the exons results in a change in the codon for Gln-2 of the signal peptide region, which introduces a translation stop signal in the downstream gene. Major sequence differences are found in the second and third introns of the apoC-I and apoC-I' genes, which contain 9 and 7.5 copies, respectively, of Alu family sequences. The apoC-I gene is expressed primarily in the liver, and it is activated when monocytes differentiate into macrophages. In contrast, no mRNA product of the apoC-I' gene can be detected in any tissue, suggesting that it may be a pseudogene. The similar structures and the proximity of the apoE and apoC-I genes suggest that they are derived from a common ancestor. Furthermore, they may be considered to be constituents of a family of seven apolipoprotein genes (apoE, -C-I, -C-II, -C-III, -A-I, -A-II, and -A-IV) that have a common evolutionary origin.

Alu RNA transcribed in vitro binds the 68-kDa subunit of the signal recognition particle.

Antibodies directed against the 68-kDa subunit of signal recognition particle (SRP) precipitate an Alu RNA X protein complex formed during in vitro transcription of a plasmid containing an Alu family sequence. The same Alu RNA X protein complex is precipitated by anti-Alu sera from certain patients with systemic lupus erythematosus and related autoimmune diseases (Kole, R., Fresco, L. D., Keene, J. D., Cohen, P. L., Eisenberg, R. A., and Andrews, P. G. (1985) J. Biol. Chem. 260, 11781-11786). Similarly to anti-SRP antibodies human, anti-Alu sera precipitate SRP from HeLa cell extract and detect a 68-kDa SRP subunit on immunoblots. These results indicate that the Alu antigen and the 68-kDa SRP subunit are the same polypeptide.

Recent progress with the DNA repair mutants of Chinese hamster ovary cells.

Repair-deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant, which is noted for its very high sister-chromatid exchange frequencies. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to chlorodeoxyuridine (CldUrd). After EcoRI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25-30 kilobases (kb) was present. Since the DNA molecules used to produce these transformants were sheared to less than 50 kb in size, the correcting gene should be small enough to clone in a cosmid vector. Using drug-resistance markers to select for hybrids after fusion, we have done complementation experiments with ultraviolet light (u.v.)-sensitive mutants and have identified a sixth complementation group, line UV61. Additionally, CHO mutants UV27-1 and MMC-2, isolated in other laboratories, were found to belong to UV group 3, which is represented by line UV24. To study the behaviour of transfected DNA molecules in repair-deficient cells, we treated plasmid pSV2gpt with either u.v. radiation or cis-diamminedichloroplatinum(II) (cis-DDP) and introduced the damaged DNA into normal CHO cells (AA8) and mutants UV4 and UV5. Unrepaired damage to the plasmid was indicated by loss of colony-forming ability of the transfected cells in selective medium containing mycophenolic acid. With u.v. damage, the differential survival of the cell lines was similar to that seen when whole cells are treated with u.v. However, with cis-DDP damage, mutant UV4 did not exhibit the extreme hypersensitivity (50-fold) that occurs when cells are treated. This result suggests that UV4 cells may be able to repair cross-links in transfected DNA.

Clustering and subfamily relationships of the Alu family in the human genome.

Thirteen and 10 sequences of the Alu family of repeated DNA elements found within the human thymidine kinase and beta-tubulin genes, respectively, were compared. These genes have approximately five times the expected density of Alu family members. The consensus sequence that could be drawn from these 23 Alu family members would differ slightly from others drawn from random Alu family sequences but only at very heterogeneous positions. The different Alu family members do show different pairwise percentage identities, with approximately 15% (7 of 48 Alu family members analyzed) of them clearly representing a separate subfamily of sequences. This analysis also confirms the species-specific differences between human and the prosimian Galago crassicaudatus Alu family members. These data are consistent with both the origin of these sequences in primates less than 65-70 Myr ago and amplification since that time to their present 500,000 copies. The data do not show any special relationships among densely clustered Alu family members.

Isolation of three kinds of human endogenous retrovirus-like sequences using tRNA(Pro) as a probe.

Three kinds of human endogenous retrovirus-like sequences (HuERS-P1, 2 and 3) were isolated from a HeLa cell genomic library using the 3'-half fragment of proline tRNA as a hybridization probe. These elements contained putative primer binding sites complementary to the 3'-terminus of proline tRNA and long terminal repeats (LTRs) characteristic of retrovirus provirus. The LTR sequence of HuERS-P1 consisted of about 690 nucleotides and contained a CAT box, a TATA box and a polyadenylation signal. A complete unit of an Alu family sequence was inserted into the 5'-LTR of one of the clones. HuERS-P2 also contained a TATA box and a polyadenylation signal in its LTR (about 840 nucleotides long), but the LTR sequence of this element was quite different from that of HuERS-P1. Although clone HuERS-P3 contained only the 5'-LTR region, this LTR sequence contained a CAT box, a TATA box and a poly-adenylation signal and was quite similar to the LTR sequence of the recently isolated human retrovirus-related sequence HuRRS-P (Kröger, B. and Horak, I. (1987) J. Virol., 61, 2071-2075). Human and simian DNAs contain 10 to 40 copies of these elements, but mouse DNA does not contain these elements.

Structure and in vitro transcription of a mouse B1 cluster containing a unique B1 dimer

A highly repetitive DNA element located 950 bp upstream from a mouse U 2 small nuclear RNA gene has been cloned and characterized. The repetitive element is composed of a simple sequence repeat and a cluster of three B1 sequences. Two of these B1 elements are arranged head-to-tail and are joined by an oligo(dA)-rich linker. This unique B1 dimer, comprised of 339 bp, resembles the dimeric structure of primate Alu-family sequences, particularly that of a prototypic human Alu element. The other B1 element within the mouse cluster is a typical monomeric unit. Transcription studies performed in HeLa cell extracts with deletion mutants of the B1 cluster reveal that the single B1 unit is expressed at least 50 times more efficiently than the Bl dimer region. Furthermore, the B1 dimer which contains mutations in the first polymerase III promoter region is not transcribed end-to-end. We conclude that this B1 dimer is unlikely to give rise to a new dimeric retroposon family in the mouse genome.

The multiple origins of human Alu sequences.

I have analyzed a collection of published human Alu sequences. The compiled sequences show several unexpected features, including a uniform pattern of divergence from their consensus sequence, a mutual divergence that is not correlated with their age, and common features in the genomic DNA flanking the 5' ends of the elements. I suggest that the Alu family of sequences derives from a large pool of precursors and not from a single precursor similar to the family consensus sequence, and that new elements integrate into the genome selectively at oligo-A-rich sites.

Replication of a plasmid bearing a human Alu-family repeat in monkey COS-7 cells.

Monkey COS-7 cells were transformed with BLUR8 DNA, a pBR322 plasmid containing a human Alu-family sequence at the BamHI site. Within 24 hr of transformation 2-5% of the BLUR8 molecules recovered resisted cleavage by Dpn I, indicating they had replicated. Electron microscopy revealed appropriately sized circular molecules with replication bubbles whose centers were mapped to the Alu insert. A 16-base-pair deletion within the Alu sequence prevented replication. The results indicate that certain Alu sequences can serve as origins of replication in COS-7 cells.

Transforming activity of human papillomavirus type 16 DNA sequence in a cervical cancer.

A genomic DNA sample from cervical cancer tissue, containing human papillomavirus (HPV) type 16, was found to induce malignant transformation of NIH 3T3 cells when it was tested by transfection assays using the calcium phosphate coprecipitation technique. The primary and secondary transformants contained the HPV type 16 DNA sequences and human specific Alu family sequences. To the best of our knowledge, it has not been reported previously that HPV type 16 DNA sequences in total genomic DNA from a cervical cancer have transforming activity.