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PubMed2005

The emerging role of the new aromatase inhibitors in the treatment of breast cancer.

The clinical development of aromatase inhibitors in recent years represents a significant addition to our armamentarium for the treatment of postmenopausal patients with hormone receptor-positive breast cancer--both for metastatic disease and in the adjuvant setting. In patients with metastatic disease, third-generation aromatase inhibitors have shown significantly superior efficacy over tamoxifen as first-line hormone therapy. In the adjuvant setting, preliminary results with the use of aromatase inhibitors in ongoing large clinical trials indicate significant gains in disease-free survival rates and in the occurrence of contralateral breast cancer, either alone for 5 years or sequentially after tamoxifen for 2-3 or 5 years. While tamoxifen monotherapy continues to be standard adjuvant therapy for patients with low risk primary breast cancer, postmenopausal patients with higher risk hormone-positive primary breast cancer should also be offered an aromatase inhibitor. Aromatase inhibitors are also prescribed for patients with contraindications to tamoxifen. The optimal sequence and duration of aromatase inhibitor adjuvant therapy as well as its long-term impact on overall survival remain to be established. In general, aromatase inhibitors have a good toxicity profile. Long-term effects such as the risk of osteoporosis need to be better defined.

Age Factors↗
PubMed1979

Mutation generating a fragment of the major heat shock-inducible polypeptide in Drosophila melanogaster.

Drosophila melanogaster tissues carrying a third chromosome with the deletion Df(3R)Kar(D2) make a 40,000-dalton (Dal) heat shock protein not made by wild type. The unusual polypeptide was inducible in every tissue examined. Tryptic peptide fingerprints showed it to include part of the 70,000-Dal major heat shock protein. Mapping experiments placed the mutation responsible for the 40,000-Dal protein at or close to the kar(D2) deletion. One break point of the deletion is in subdivision 87A, close to or at a heat shock locus that codes for the 70,000-Dal protein. The results are consistent with the possibility that this break point is within a gene for the 70,000-Dal protein, leaving only the initial portion of its coding sequence. This would specify the direction of transcription of the mutant gene as proximal to distal on the normal chromosome. The 87A heat shock locus should contain at least two genes for the 70,000-Dal protein, because embryos homozygous for the kar(D2) deletion and lacking the heat shock locus at 87C, which also codes for the 70,000-Dal protein, nevertheless produced both the 40,000-Dal and the 70,000-Dal proteins upon temperature elevation. Using the presence of the 40,000-Dal protein to monitor chromosome segregation, we found that embryos homozygous for deletions of the heat shock puff site at 93D exhibited a normal electrophoretic pattern of heat shock proteins.

Animals↗
PubMed2002

Phylogeography of endemic ermine (Mustela erminea) in southeast Alaska.

The North Pacific Coast (NPC) of North America is a region of high mammalian endemism, possibly due to its highly fragmented landscape and complex glacial history. For example, four island and one mainland subspecies of ermine, Mustela erminea, have been described as endemic to southeast Alaska alone. To better understand the role of past climatic change in generating diversity in the region, we examined DNA sequence variation in the mitochondrial cytochrome b gene of 210 ermine from across North America, with an emphasis on Alaska and British Columbia. We found three distinct (1.5-3.6% uncorrected 'p') lineages of ermine, all of which occur in southeast Alaska. One lineage includes a southeast Alaska endemic and specimens from Alaska (outside of southeast) and Eurasia. A second lineage includes two southeast Alaskan endemics and ermine from western Canada and the coterminous United States. The close relationships of these purported endemics to ermine outside of southeast Alaska suggest that they colonized the region from Beringian and southern glacial refugia, respectively, following deglaciation of the NPC. The third lineage appears restricted to the Prince of Wales Island complex in southeast Alaska (two subspecies) and Graham Island (Haida Gwaii), British Columbia. This restricted distribution suggests that these populations may be derived from relicts that persisted in a coastal refugium during the Wisconsin glaciation. Studies of nuclear genes and adaptive morphological evolution are necessary to further explore discrepancies between the geographical pattern of differentiation based on mtDNA and the existing subspecific taxonomy based on morphology.

Alaska↗
PubMed1993

Sorbitol dehydrogenase. Full-length cDNA sequencing reveals a mRNA coding for a protein containing an additional 42 amino acids at the N-terminal end.

A cDNA clone encoding rat sorbitol dehydrogenase (SDH) was isolated from a rat testis lambda ZAP II cDNA library. The full-length cDNA insert contained 2277 base pairs (bp), starting 182 bp upstream from an ATG codon where translation to the active enzyme SDH is presumed to be initiated. A second ATG codon, however, was found 126 bp upstream, aligned in the same reading frame as that of the active enzyme. Therefore, the coding sequence for SDH can be translated into an additional 42-amino-acid polypeptide linked to the N-terminal amino acid of the enzyme, generating a pre-sorbitol dehydrogenase. The sequence data indicate that the nucleotide environment around this ATG codon is more favorable towards it being the actual open reading frame (ORF) for a pre-SDH than the ATG codon preceding the nucleotide sequence for SDH. Since no known SDH starts with the additional 42 amino acids, it may be that post-translational removal of this polypeptide accompanies the release of the active enzyme. Next, the 3' untranslated region of the cDNA contained a non-coding 1021 bp downstream from the TAA stop codon. The latter sequence included three putative poly(A) signals: one at nucleotides 1362-1367, the second at nucleotides 1465-1470, and the third at nucleotides 2212-2217 [17 bp away from the poly(A) tail]. In addition to the above findings we also report a variance in one of the amino acids in the SDH cDNA sequence. This variance occurs at position 957-960, where threonine is coded for instead of aspartic acid; in the rat testis SDH cDNA, we find the sequence is ACG instead of GAC, as was reported for the rat liver SDH cDNA. Northern-blot hybridization analysis showed that SDH mRNA is a doublet, one band of 4 kb and the other of 2.3-2.4 kb, in both the rat liver and the rat lens, further confirming that the isolated SDH cDNA constituted a full-length cDNA.

Amino Acid Sequence↗
PubMed2005

Malaria in the post-genomics era: light at the end of the tunnel or just another train?

Malaria remains the third leading cause of death attributable to an infectious disease worldwide, with an estimated death toll of over 2 million per year, predominately in sub-Saharan Africa. The first serious attempt to eradicate this disease was unsuccessful, and 50 years later in 1998 a second programme coined "roll back malaria" was started. While this programme is at present unlikely to reach its stated aims, the completion of the genome sequencing projects on the human host, the mosquito vector, and the malaria parasite offers new hope. It is probable that the burden of disease caused by the most malignant form of the parasite Plasmodium falciparum can be, if not eliminated, then effectively suppressed within a generation through new and novel treatments aimed at all three arms of malaria control.

Animals↗
PubMed1988

Sequence of centromere separation: generation of unstable multicentric chromosomes in a rat cell line.

A transformed cell line, B1, of cerebral endothelial origin from the Wistar-Kyoto male rat has chromatid and chromosome type bridges in virtually every cell. It exhibits various dicentric and polycentric chromosomes. Most dicentrics are symmetric isochromosomes. Certain isodicentrics are present in a fair segment of the cell population; however, almost all cells have some newly arising isodicentrics. The live cells show a lengthened prometaphase. Anaphase is also retarded possibly due to the occurrence of bridges. At anaphase some multicentrics split at only one centromere. When pulled to the two poles the unsplit centromeres and the distal chromosome segment form a side arm bridge. Another mechanism appears to be a total lack of separation of daughter centromeres at meta-anaphase ('meiotic-like' behavior of mitotic chromosomes). This is realized by the pulling of each of the two unsplit centromeres to opposite poles and results in bridges with both sister chromatids running parallel to each other. A break at corresponding weak points in the two sister chromatids followed by rejoining can form a dicentric isochromosome. A third mechanism, the breakage-fusion-bridge cycle, is also operative but would not produce isodicentrics. In the case of the first two mechanisms some or all centromeres apparently split between telophase and onset of the following DNA synthesis rather than at the usual time at late metaphase. These observations may suggest some previously unknown behavior of multicentric chromosomes during mitosis.

Anaphase↗
PubMed2003

Beta-synuclein inhibits formation of alpha-synuclein protofibrils: a possible therapeutic strategy against Parkinson's disease.

Parkinson's disease (PD) is an age-associated and progressive movement disorder that is characterized by dopaminergic neuronal loss in the substantia nigra and, at autopsy, by fibrillar alpha-synuclein inclusions, or Lewy bodies. Despite the qualitative correlation between alpha-synuclein fibrils and disease, in vitro biophysical studies strongly suggest that prefibrillar alpha-synuclein oligomers, or protofibrils, are pathogenic. Consistent with this proposal, transgenic mice that express human alpha-synuclein develop a Parkinsonian movement disorder concurrent with nonfibrillar alpha-synuclein inclusions and the loss of dopaminergic terminii. Double-transgenic progeny of these mice that also express human beta-synuclein, a homologue of alpha-synuclein, show significant amelioration of all three phenotypes. We demonstrate here that beta- and gamma-synuclein (a third homologue that is expressed primarily in peripheral neurons) are natively unfolded in monomeric form, but structured in protofibrillar form. Beta-synuclein protofibrils do not bind to or permeabilize synthetic vesicles, unlike protofibrils comprising alpha-synuclein or gamma-synuclein. Significantly, beta-synuclein inhibits the generation of A53T alpha-synuclein protofibrils and fibrils. This finding provides a rationale for the phenotype of the double-transgenic mice and suggests a therapeutic strategy for PD.

Amino Acid Sequence↗
PubMed1992

Modulation of DNA binding specificity by alternative splicing of the Wilms tumor wt1 gene transcript.

The technique of whole-genome polymerase chain reaction was used to study the DNA binding properties of the product of the wt1 gene. The zinc finger region of this gene is alternatively spliced such that the major transcript encodes a protein with three extra amino acids between the third and fourth fingers. The minor form of the protein binds specifically to DNA. It is now shown that the major form of wt1 messenger RNA encodes a protein that binds to DNA with a specificity that differs from that of the minor form. Therefore, alternative splicing within the DNA binding domain of a transcription factor can generate proteins with distinct DNA binding specificities and probably different physiological targets.

Amino Acid Sequence↗
PubMed2000

Genetic analysis of the requirements for alpha-actinin function.

Null alpha-actinin mutations in Drosophila are lethal and produce conspicuous defects in muscle structure and function. Here, we used transgene rescue to examine the requirements for alpha-actinin function in vivo. First, we tested the ability of a cDNA-based transgene encoding the adult muscle isoform of alpha-actinin under control of the heterologous ubiquitin promoter to rescue the lethality of null alpha-actinin mutations. Successful rescue indicated that alternative splicing, which also generates larval muscle and non-muscle isoforms, was not essential for viability and that there were no strict spatial or temporal requirements for alpha-actinin expression. Secondly, chimeric transgenes, with functional domains of alpha-actinin replaced by similar domains from spectrin, were tested for their ability to rescue alpha-actinin mutants. Replacement of either the actin binding domain or the EF hand calcium binding domain yielded inactive proteins, indicating that these conserved domains were not functionally equivalent. Thirdly, the length of alpha-actinin was modified by adding a 114 amino acid structural repeat from alpha-spectrin to the center of the rod domain of alpha-actinin. Addition of this sequence module was expected to increase the length of the native alpha-actinin molecule by at least 15%. yet was fully compatible with alpha-actinin function as measured by rescued lethality and flight. Thus, unexpectedly, the exact length of alpha-actinin was not critical to its function in the muscle Z disk.

Actinin↗
PubMed1996

Generation of mice with a 200-kb amyloid precursor protein gene deletion by Cre recombinase-mediated site-specific recombination in embryonic stem cells.

Gene disruptions and deletions of up to 20kb have been generated by homologous recombination with appropriate targeting vectors in murine embryonic stem (ES) cells. Because we could not obtain a deletion of about 200 kb in the mouse amyloid precursor protein gene by the classical technique, we employed strategies involving the insertion of loxP sites upstream and downstream of the region to be deleted by homologous recombination and elicited excision of the loxP-flanked region by introduction of a Cre expression vector into the ES cells. In the first approach, the loxP sequences were inserted in two successive steps and after each step, ES cell clones were isolated and characterized. Deletion of the loxP-flanked sequence was accomplished by introducing the cre gene in a third step. In the second approach, ES cells containing the upstream loxP cassette were electroporated simultaneously with the downstream loxP targeting vector and the Cre expression plasmid. ES cells were obtained that gave rise to chimeric mice capable of germ-line transmission of the deleted amyloid precursor protein allele.

Amino Acid Sequence↗
PubMed1998

Generation of switch hybrid DNA between Ig heavy chain-mu and downstream switch regions in B lymphocytes.

Ig heavy chain isotype switching in B lymphocytes is known to be preceded by transcription of a portion of the particular heavy chain gene segment that is targeted for recombination. Here, we describe an active role for these transcripts in the switch recombination process. Using an in vitro assay that exposes an artificial switch-mu (Smu) minisubstrate to switch region transcripts in the presence of nuclear extracts from switching cells, we demonstrate that free 3' ends of the Smu sequence are extended onto switch region transcripts by reverse transcription. The activity was induced in splenic B lymphocytes upon activation with LPS or CD40 ligand. This in vitro process is thought to be relevant to in vivo class switching for two reasons: 1) although only one-third of the Smu minisubstrate actually contains Smu sequence, all crossovers between switch regions occurred in the Smu portion; and 2) treatment of B lymphocytes with IL-4, which enriches for switching to S gamma 1, increases the ratio of Smu-S gamma 1 to Smu-S gamma 3 hybrids by 16% after LPS treatment and by 37% after CD40 ligand activation, implicating this S mu-primed reverse transcription of switch region transcripts as a novel mechanism of regulating the specificity of isotype switching. Further evidence for an active role of switch region transcripts was obtained by expressing S alpha RNA in trans in the Bcl1B1 B lymphoma line. Endogenous S mu-S alpha switch circles were detected in Bcl1B1 cells expressing exogenous S alpha RNA but not in mock-transfected cells.

Animals↗
PubMed2001

An ins(X;11)(q24;q23) fuses the MLL and the Septin 6/KIAA0128 gene in an infant with AML-M2.

The MLL (HRX, ALL-1 HTRX) gene at chromosome band 11q23 frequently is rearranged in acute lymphoblastic and myeloblastic leukemia. To date, more than 40 different 11q23 abnormalities have been described on the cytogenetic level, and at least 25 of the respective fusion partner genes are cloned. The vast majority of the respective reciprocal translocations generate a chimeric 5'-MLL/partner-3' gene on the derivative 11q23. In this work, we report a unique ins(X;11)(q24;q23) in an infant with acute myeloid leukemia (AML-M2) that fuses the human KIAA0128 gene at Xq24 with MLL. In contrast to the typical reciprocal MLL translocations, however, we provide evidence that the 5'-MLL/KIAA0128-3' fusion resides on Xq24 rather than on 11q23. The KIAA0128 gene encodes the human Septin 6 protein, which contains an ATP-GTP binding motif and three nuclear targeting sequences in its carboxy terminus. The maintenance of the reading frame of the 5'-MLL/KIAA0128-3' mRNA fusion allows for the formation of a novel chimeric protein. Septin 6 is the third member of the Septins that is fused to the MLL protein; the other two are hCDCrel at 22q11 and MSF at 17q25.

Chromosomes, Human, Pair 11↗
PubMed1994

Identification of spliced RNA species of Drosophila melanogaster gypsy retrotransposon. New evidence for retroviral nature of the gypsy element.

We have identified a novel RNA species of Drosophila melanogaster gypsy retrotransposon that is ca. 2 kb in length and corresponds to the third open reading frame (ORF3) of the gypsy element. This RNA is generated by splicing of the primary gypsy transcript, as is the case for retroviral env gene expression. Therefore, the striking resemblance between gypsy and retroviruses has now been extended by this study to the expression strategies of these retroelements. The primary structure of spliced RNA was determined, and its analysis shows that both gypsy subfamilies (6K and 7K) apparently are able to encode functionally active ORF3 translation products.

Animals↗
PubMedDate not supplied

Genomic organization of the genes encoding mouse T-cell receptor alpha-chain.

The vertebrate immune system uses two kinds of antigen-specific receptors, the immunoglobulin molecules of B cells and the antigen receptors of T cells. T-cell receptors are formed by a combination of two different polypeptide chains, alpha and beta (refs 1-3). Three related gene families are expressed in T cells, those encoding the T-cell receptor, alpha and beta, and a third, gamma (refs 4-6), whose function is unknown. Each of these polypeptide chains can be divided into variable (V) and constant (C) regions. The V beta regions are encoded by V beta, diversity (D beta) and joining (J beta) gene segments that rearrange in the differentiating T cell to generate V beta genes. The V gamma regions are encoded by V gamma, J gamma and, possibly, D gamma gene segments. Studies of alpha complementary DNA clones suggest that alpha-polypeptides have V alpha and C alpha regions and are encoded by V alpha and J alpha gene segments and a C alpha gene. Elsewhere in this issue we demonstrate that 18 of 19 J alpha sequences examined are distinct, indicating that the J alpha gene segment repertoire is much larger than those of the immunoglobulin (4-5) or beta (14) gene families. Here we report the germline structures of one V alpha and six J alpha mouse gene segments and demonstrate that the structures of the V alpha and J alpha gene segments and the alpha-recognition sequences for DNA rearrangement are similar to those of their immunoglobulin and beta-chain counterparts. We also show that the J alpha gene-segment organization is strikingly different from that of the other immunoglobulin and rearranging T-cell gene families. Eighteen J alpha gene segments map over 60 kilobases (kb) of DNA 5' to the C alpha gene.

Amino Acid Sequence↗
PubMed2004

Establishing an efficient Ac/Ds tagging system in rice: large-scale analysis of Ds flanking sequences.

A two-element Activator/Dissociation (Ac/Ds) gene trap system was successfully established in rice (Oryza sativa ssp. japonica cv. Nipponbare) to generate a collection of stable, unlinked and single-copy Ds transposants. The germinal transposition frequency of Ds was estimated as an average of 51% by analyzing 4413 families. Study of Ds transposition pattern in siblings revealed that 79% had at least two different insertions, suggesting late transposition during rice development. Analysis of 2057 Ds flanking sequences showed that 88% of them were unique, whereas the rest within T-DNA. The insertions were distributed randomly throughout the genome; however, there was a bias toward chromosomes 4 and 7, which had two times as many insertions as that expected. A hot spot for Ds insertions was identified on chromosome 7 within a 40-kbp region. One-third of Ds flanking sequences was homologous to either proteins or rice expressed sequence tags (ESTs), confirming a preference for Ds transposition into coding regions. Analysis of 200 Ds lines on chromosome 1 revealed that 72% insertions were found in genic region. Anchoring of more than 800 insertions to yeast artificial chromosome (YAC)-based EST map showed that Ds transposes preferentially into regions rich in expressed sequences. High germinal transposition frequency and independent transpositions among siblings show that the efficiency of this system is suitable for large-scale transposon mutagenesis in rice.

Arabidopsis↗
PubMed2002

Functional and comparative bioinformatic analysis of expressed genes from wheat spikes infected with Fusarium graminearum.

Fusarium head blight, caused by the fungus Fusarium graminearum, is a major disease on wheat (Triticum aestivum L.). Expressed sequence tags (ESTs) were used to identify genes expressed during the wheat-F. graminearum interaction. We generated 4,838 ESTs from a cDNA library prepared from spikes of the partially resistant cultivar Sumai 3 infected with F. graminearum. These ESTs were composed of 2,831 singlet (single-copy transcripts) and 715 contigs (multiple-copy transcripts) for a total of 3,546 non-redundant sequences. Four sets of nonredundant sequences were identified. One set contains numerous, common biotic and abiotic stress-related genes. Many of these stress-related genes were represented by multiple ESTs, indicating that they are abundantly expressed. A second set comprised 16 nonredundant sequences from F. graminearum that may be required for pathogenicity. A subset of these fungal genes encodes proteins associated with plant cell wall degradation. A third set of 326 nonredundant sequences had no DNA or amino acid sequence similarity to almost 1 million plant and over 7 million animal sequences in dbEST (as of 22 June 2001). Thus, these 326 nonredundant sequences have only been found in our F. graminearum-infected 'Sumai 3' cDNA library. A fourth set of 29 nonredundant sequences was found in our F. graminearum-infected wheat and another plant-pathogen interaction cDNA library. Some of these sequences encode proteins that may act in establishing various plant-fungal interactions.

Cell Wall↗
PubMed2006

One-class-at-a-time removal sequence planning method for multiclass classification problems.

Using dynamic programming, this work develops a one-class-at-a-time removal sequence planning method to decompose a multiclass classification problem into a series of two-class problems. Compared with previous decomposition methods, the approach has the following distinct features. First, under the one-class-at-a-time framework, the approach guarantees the optimality of the decomposition. Second, for a K-class problem, the number of binary classifiers required by the method is only K-1. Third, to achieve higher classification accuracy, the approach can easily be adapted to form a committee machine. A drawback of the approach is that its computational burden increases rapidly with the number of classes. To resolve this difficulty, a partial decomposition technique is introduced that reduces the computational cost by generating a suboptimal solution. Experimental results demonstrate that the proposed approach consistently outperforms two conventional decomposition methods.

Algorithms↗
PubMed1999

A direct estimate of the human alphabeta T cell receptor diversity.

Generation and maintenance of an effective repertoire of T cell antigen receptors are essential to the immune system, yet the number of distinct T cell receptors (TCRs) expressed by the estimated 10(12) T cells in the human body is not known. In this study, TCR gene amplification and sequencing showed that there are about 10(6) different beta chains in the blood, each pairing, on the average, with at least 25 different alpha chains. In the memory subset, the diversity decreased to 1 x 10(5) to 2 x 10(5) different beta chains, each pairing with only a single alpha chain. Thus, the naïve repertoire is highly diverse, whereas the memory compartment, here one-third of the T cell population, contributes less than 1 percent of the total diversity.

Adult↗