bluuebunny/crossref_metadata_2025 huggingface dataset:
GrailEXP and Genome Analysis Pipeline for Genome Annotation
Authors: Uberbacher Edward C. , Hyatt Doug , Shah Manesh
Published: June 2004

The Gene Recognition and Analysis Internet Link (GRAIL) is one of the most widely used systems for evaluating the protein‐coding potential of anonymous DNA sequences. This unit describes the use of the XGRAIL and genQuest client‐server applications to locate exons in DNA sequences, to develop gene models, and to search databases for homologs. A support protocol describes how to obtain the GRAIL and genQuest client software by anonymous FTP.
Detection of Mutations by Single‐Strand Conformation Polymorphism (SSCP) Analysis and SSCP‐Hybrid Methods
Authors: Warren William , Hovig Eivind , Smith‐Sørensen Birgitte , Børresen Anne‐Lise , Fujimura Frank K. , Liu Qiang , Feng Jinong , Sommer Steve S.
Published: June 2004

Single‐strand conformation polymorphism (SSCP) analysis detects mutations based on the fact that single‐nucleotide changes in DNA sequences alter the mobility of single‐stranded DNA in nondenaturing gels. Four methods for detecting mutations based on SSCP are described here. (1) Traditional SSCP analysis is technically easy and can be used for screening large numbers of samples. SSCP‐hybrid methods detect mutations based on either an SSCP effect or an altered component independent of the SSCP effect. (2) Dideoxy fingerprinting (ddF) involves PCR amplification of the target and creation of a set of dideoxy‐terminated strands with the mutation. (3) Bi‐directional dideoxy fingerprinting (Bi‐ddF) involves production of two sets of dideoxy‐terminated strands that are generated from two different primers. (4) Restriction endonuclease fingerprinting (REF) involves cleavage of the amplified target with five to six groups of restriction endonucleases.
Chemical Cleavage of Heteroduplex DNA to Identify Mutations
Authors: Cotton Richard G. H. , Grompe Markus
Published: June 2004

Mutation screening by the chemical‐cleavage method is based on the fact that mismatched cytosine (C) and thymidine (T) are more reactive with the compounds hydroxylamine and osmium tetroxide, respectively, than are Watson and Crick‐paired cytosine and thymidine bases. In this protocol, an excess of unlabeled target DNA is hybridized with labeled wild‐type DNA probe and heteroduplexes are formed. One aliquot is treated with hydroxylamine, which reacts with mismatched C bases. Another aliquot is treated with osmium tetroxide, which reacts with mismatched T bases. The reactions are mixed with piperidine; the strands are then cleaved at the sites where hydroxylamine and osmium tetroxide react. Cleaved fragments are then electrophoresed and sized on polyacrylamide gels, identifying the point of cleavage (and hence the position of the mutation). Then only a small portion of the mutant gene needs to be sequenced to define a single change between two DNA sequences.
Mutation Detection by Cycle Sequencing
Authors: Thierfelder Ludwig
Published: June 2004

Candidate genes are screened for mutations by a DNA sequencing procedure known as cycle sequencing. First, a segment of the candidate gene is PCR amplified from the genomic DNA of an affected individual. The PCR product is then subjected to multiple rounds of further amplification in a thermal cycler using a heat‐stable DNA polymerase in the presence of different dideoxynucleotides and a radiolabeled primer. The resulting 32P‐labeled sequence reaction products are fractionated on a denaturing polyacrylamide gel and visualized by autoradiography. DNA segments on the order of 200 bp from 10 to 30 individuals can be screened on each gel. Cycle sequencing eliminates the need to subclone genomic fragments or PCR products, which makes it a much simpler method than conventional sequencing for identifying mutations.
Heterozygote Detection Using Automated Fluorescence‐Based Sequencing
Authors: Dabora Sandra L. , Arad Michael , Barr Scott , Kim Jae Bum
Published: June 2004

Point mutations are the most common type of polymorphism. These mutations can occur in one allele, while the other allele retains the normal sequence; detecting such sequence variants in a heterozygous individual's DNA, carrying both the normal allele and the mutant allele, can be difficult. In this unit, a strategy is presented for detecting such sequence variation using automated fluorescence‐based sequencing traces, and analysis with software tools that assist with the identification of sequence variation. A protocol is given that has been used to identify heterozygote mutations in TSC1 and TSC2, the causative genes for the autosomal dominant disorder tuberous sclerosis complex (TSC), as well as for a number of other inherited disorders in humans. Common genetic diseases like hypertrophic cardiomyopathy (HCM) are caused by autosomal dominant mutations in a subset of sarcomere proteins. An alternate protocol describes an approach for large‐scale sequencing adapted for serial screening for HCM mutations.
DNA Mutation Detection Using Denaturing High‐Performance Liquid Chromatography (DHPLC)
Authors: Oefner Peter J. , Underhill Peter A.
Published: June 2004

This method permits the rapid detection of singlebase substitutions and insertions or deletions of up to 1.5 kb in PCRamplified DNA fragments. The method exploits the differential retention of homo and heteroduplex DNA species under conditions of partial thermal denaturation and provides an efficient method to rapidly compare DNA fragments for sequence variation, once this variation has been identified by conventional sequencing.
Mutation Nomenclature
Authors: den Dunnen Johan T. , Antonarakis Stylianos E.
Published: June 2004

As part of the Human Genome Variation Society (formerly known as the HUGO Mutation Database Initiative), a committee was formed to suggest standards for the description of sequence variants in DNA, RNA, and protein sequences. The committee proposed that the nomenclature should be unequivocal, precise, and short, and should prevent any possible confusion and follow existing practice as much as possible. To “spread the word,” the nomenclature rules were published at regular intervals. This unit summarizes these nomenclature recommendations, which stimulated a uniform and unequivocal description of sequence variants in literature.
Quality Assurance and Quality Control in Clinical Cytogenetics
Authors: Watson Michael S.
Published: June 2004

The goal of any clinical laboratory should be to provide patients with the most accurate test results possible. This is accomplished through various overlapping programs that continuously monitor and optimize all aspects of a test, including the decision to request the test, the test itself, and the reporting of results to the referral source and patient. The levels at which test performance and accuracy can be optimized are encompassed under quality control (QC) and quality assurance (QA). This unit reviews quality control and assurance guidelines, in addition to discussing how to establish a quality assurance program.
Preparation of Metaphase Spreads from Chorionic Villus Samples
Authors: Jackson Laird , Gibas Longina M. , Barr Marie A.
Published: June 2004

Chorionic villi are composed of an outer layer of trophoblastic cells and an inner mesenchymal cell core, both of fetal origin. Cytogenetic analysis of chorionic villi can be accomplished using material prepared in either of two ways. In the culture method described in this unit, villi are disaggregated by mechanical and enzymatic methods, and the resulting cell suspension is used to establish primary cultures. Mesenchymal cells of the villus core are released by this procedure and the fibroblasts are actively proliferative in tissue culture. Cultures can be used for cytogenetic analysis after ˜1 week. In the “direct” technique, presented here in an , Langhans cells of the cytotrophoblast, actively dividing cells in first‐trimester villi, are synchronized and arrested in mitosis after a short incubation period, and metaphase spreads are prepared.Chorionic villi are composed of an outer layer of trophoblastic cells and an inner mesenchymal cell core, both of fetal origin
Analysis of Sister‐Chromatid Exchanges
Authors: German James , Alhadeff Becky
Published: June 2004

Two requirements for the cytogenetic analysis of sister‐chromatid exchanges (SCEs) in somatic cells are (1) a population of actively proliferating cells that will provide an adequate number of metaphases and (2) sister chromatids that in some way are differentially labeled or stained in the metaphases. SCEs can be recognized as abrupt discontinuities in the staining patterns of the two chromatids of a metaphase chromosome at what appear to be identical sites, with reciprocal switching from one chromatid to its sister. This protocol uses phytohemagglutinin (PHA)‐stimulated cultures of blood lymphocytes as a source of proliferating cells. The cells are incubated with the thymidine analog BrdU. Slides prepared from fixed cells with BrdU‐substituted chromosomes are treated with Hoechst 33258, exposed to light and heat, and then Giemsa‐stained to produce differentially stained chromosomes. The chromatids with bifilar substitution exhibit a lighter purple stain than their unifilarly substituted sister chromatids.
Determination of Chromosomal Aneuploidy Using Paraffin‐Embedded Tissue
Authors: Schofield Deborah E.
Published: June 2004

Tissue processed in pathology departments is generally fixed in formalin and embedded in paraffin blocks. Depending upon the nature of the question asked, two different approaches are taken to analyze this material and are presented in this unit. If information about the spatial relationship of one cell to another is not necessary, a thick section of tissue can be disaggregated and the resulting intact nuclei analyzed. The number of hybridization signals per nucleus is tabulated; variance from two hybridization signals per nucleus indicates aneuploidy. If the spatial relationship of one cell to another needs to be preserved, intact tissue sections on glass slides can be analyzed. Tissue processed in pathology departments is generally fixed in formalin and embedded in paraffin blocks.
Overview of Molecular Genetic Diagnosis
Authors: Korf Bruce R. , Pagon Roberta A.
Published: June 2004

This unit describes the two approaches for molecular genetic diagnosis, direct mutation analysis and linkage studies. Direct mutation analysis is possible if the gene responsible for a disorder has been cloned and techniques exist to detect mutations. Genetic linkage analysis is used when polymorphic markers are available that are closely linked to a disease gene but the gene itself has not been cloned, or when it is difficult or impossible to detect mutations in the gene. For direct mutation analysis, the unit offers a review of the types of mutations that can be detected and the strategies that can be employed for their detection. The unit then describes the types of disorders that are best suited for linkage‐based analysis and offers guidance for interpreting the data.
Quality Assurance and Quality Control in Diagnostic Molecular Biology
Authors: Murphy Patricia D.
Published: June 2004

The newest forms of clinical diagnostic testing are based upon tools gleaned from molecular biology. Manipulations of RNA and DNA from various tissue sources (e.g., skin, blood, urine, excised tumor) can provide valuable information about an individual's genetic status or the basis or potential for metastasis of a tumor. As of 2002, over 200 laboratories in the United States provide clinical services. Particularly when such information is to be used to provide genetic counseling or determine treatment, care must be taken to ensure accuracy in the generation and reporting of results. This unit outlines the aspects of quality assurance that are important in nucleic acid‐based diagnostic testing. More specifically, it describes the three elements of a quality assurance program: the directors qualifications, on‐site laboratory inspection, and interlaboratory comparison and proficiency testing. The unit also discusses how to evaluate if a particular genetic test is suitable for clinical testing.
Single‐Cell DNA and FISH Analysis for Application to Preimplantation Genetic Diagnosis
Authors: Chong Samuel S. , Gore‐Langton Robert E. , Hughes Mark R. , Miron Patricia Minehart
Published: June 2004

The goal of preimplantation genetic diagnosis (PGD) is to avoid transfer of embryos affected with a specific genetic disease or condition. This unit describes the steps involved in amplifying DNA from a single blastomere and specific assays for detecting a variety of DNA mutations. For some assays, whole‐genome amplification by primer‐extention preamplification (PEP) is performed prior to analysis. Support protocols describe the biopsy of one or two blastomeres from the developing preimplantation embryo, isolation for further investigation of all blastomeres from embryos shown to have the mutant allele, and isolation of single lymphocytes or lymphoblastoid cells as models for single‐cell DNA analysis. A procedure for FISH analysis on single interphase blastomeres is provided along with a support protocol for probe validation that is recommended as a preliminary step before performing any PGD FISH analysis.
Searching Online Mendelian Inheritance in Man (OMIM) for Information for Genetic Loci Involved in Human Disease
Authors: Baxevanis Andreas D.
Published: June 2004

Online Mendelian Inheritance in Man (OMIM) is a non‐sequence‐based information resource that can be of tremendous use to genomics researchers, physicians, and patients. There are two main ways in which a user can search the OMIM database. One may choose to search the OMIM database directly from the NCBI home page, as described in this unit. Alternatively, OMIM can be downloaded and run on any internal site where the user may want to keep from submitting data across the Web or where a local installation would be otherwise advantageous. describes how to navigate through an OMIM record. Online Mendelian Inheritance in Man (OMIM) is a non‐sequence‐based information resource that can be of tremendous use to genom Mendelian Inheritance in Man (OMIM) is a non‐sequence‐based information resource that can be of tremendous use to genomics researchers, physicians, and patients.
Overview of Genetic Diagnosis in Cancer
Authors: Korf Bruce R.
Published: June 2004

Both cytogenetic and molecular genetic studies can contribute to the management of patients with cancer. In some cases genetic markers are specific to particular tumor types and are useful in diagnosis. This can be helpful in distinguishing histologically similar tumors that may respond differently to treatment and can sometimes be of prognostic value. Genetic markers can also be tools for following the response of a tumor to therapy, providing a sensitive means of detection of relapse. This introductory unit considers some of the types of genetic changes that occur in association with malignancies and major approaches to their detection.
Metaphase Harvest and Cytogenetic Analysis of Solid Tumor Cultures
Authors: Fletcher Jonathan
Published: June 2004

Cytogenetic preparations of solid tumor specimens often demonstrate clonal chromosome aberrations. These clonal aberrations are useful in confirming the neoplastic nature of a given tumor and might also suggest a specific diagnosis or prognosis. Most solid tumors must be cultured for several days before metaphase cells can be obtained for cytogenetic analysis. Thedescribes harvesting of metaphase cells from cultures prepared from solid tumor specimens. The cells are fixed and then stained using a trypsin‐Giemsa (GTG) method to produce characteristic banding patterns for cytogenetic analysis. The firstdescribes disaggregation and short‐term tissue culture of solid tumors. The seconddescribes analysis of cells from lymph nodes infiltrated by lymphoma cells and the thirddescribes the direct analysis of cells from malignant effusion (fluid) samples.
Molecular Analysis of DNA Rearrangements in Leukemias and Non‐Hodgkin's Lymphomas
Authors: Longtine Janina , Fox Edward , Reynolds Carol , Sklar Jeffrey
Published: June 2004

Genetic markers for leukemias and lymphomas include chromosomal translocations and antigen‐receptor gene rearrangements. Clonal rearrangements of immunoglobulin or T cell receptor (TCR) genes reflect clonal proliferations of lymphocytes, a characteristic feature of lymphoid neoplasia. These rearrangements can be detected as described in this unit by Southern blot hybridization or, in many instances, the polymerase chain reaction (PCR). Specific chromosomal translocations can also serve as markers for clonality, for malignant transformation, and for various defined subtypes of hematopoietic cancers. PCR protocols are described for detection of the two most commonly assayed translocations, t(9;22) of chronic myelogenous leukemia or acute lymphoblastic leukemia, and t(14;18) of follicular lymphomas.
Molecular Analysis of Gene Amplification in Tumors
Authors: Wasson Jonathon C. , Brodeur Garrett M.
Published: June 2004

Detection of gene amplification in human cancer cells has both clinical and biological importance. Amplified genes can be classified in one of two categoriesas oncogenes or as genes conferring drug resistance. Both types of gene amplification may alter clinical management of the patient. The basic protocol describes preparation and quantitation of DNA from tumor tissue and the use of conventional Southern blot hybridization analysis to detect and quantify gene amplification. The first alternate protocol provides an approach to quickly screen tumor samples for gene amplification using slot blot hybridization analysis. The second alternate protocol describes the use of the polymerase chain reaction (PCR) for analyzing tumors that may be difficult to analyze because of degradation or limited amounts of DNA. A explains the proper methods for obtaining, processing, storing, and shipping tumor tissue for DNA analysis.
Differential Display of mRNA by PCR
Authors: Livesey F.J. , Hunt S.P.
Published: June 2004

This unit outlines the polymerase chain reaction (PCR)‐based technique of mRNA differential display, which identifies genes that are differentially expressed between cells or tissues. The basic protocol describes the actual differential display PCR reaction along with details of the identification, reamplification, and cloning of candidate differentially expressed genes. A support protocol provides instructions on removing contaminating genomic DNA from the RNA samples and reverse transcribing the purified RNA to produce the cDNA used in the subsequent PCR reactions.
One‐Step Enzymatic Purification of PCR Products for Direct Sequencing
Authors: Bum Kim Jae , Blackshaw Seth
Published: June 2004

The polymerase chain reaction (PCR) has become a powerful and widespread method for analyzing DNA. This protocol offers convenient enzymatic purification of PCR products for direct sequencing without the need for further subcloning, precipitation, or column purification. All reactions may be performed in 96‐well PCR plates and are compatible with large‐scale sequencing. This method is designed to be convenient and rapid. It is suitable for a variety of PCR templates, including plasmid, lambda, and genomic DNA.
Production of Pseudotype‐Retroviral Vectors
Authors: Yee Jiing‐Kuan
Published: June 2004

Retrovirus pseudotype is defined as the genome of one retrovirus encapsidated by the envelope protein of a second virus. The host range of the pseudotype is that of the virus donating the envelope protein. Two procedures that use 293GP cells, which are derived from human kidney 293 cells, are described here. The first is based on the high transient transfection efficiency of 293 cells. The retroviral construct and an expression plasmid for VSV‐G are co‐transfected into 293GP cells that stably express MLV gag and pol proteins. Transiently generated virus is then harvested during consecutive days following DNA transfection. The second procedure involves stable 293GP cell lines containing the VSV‐G gene under the control of a promoter whose activity is regulated by tetracycline. Cell lines containing the retroviral vector of interest are established under noninduced conditions. Infectious virus can be harvested following the induction of VSV‐G expression in these cell lines.Retrovirus pseudotype is defined as the genome of one retrovirus encapsidated by the envelope protein of a second virus.
Production of Recombinant Adeno‐Associated Viral Vectors
Authors: Haberman Rebecca A. , Kroner‐Lux Gabriele , Samulski Richard Jude
Published: June 2004

Recombinant adeno‐associated viral (rAAV) vectors are capable of delivering genes to both dividing and nondividing cells. This property, along with the physical stability of the virions, permits transduction of cells both in tissue culture and in the whole animal. In this unit, protocols for the adenovirus‐free production of rAAV vectors, their purification, and their quantitation are provided. The production and purification of the rAAV vectors are described, using two different methods. Two assays for determining rAAV titers are also presented: dot blot and in vitro infection with a transgene expression assay.
Construction of Replication‐Defective Herpes Simplex Virus Vectors
Authors: Goins William F. , Marconi Peggy , Krisky David , Wolfe Darren , Glorioso Joseph C. , Ramakrishnan Ramesh , Fink David J.
Published: June 2004

Advances in identification and characterization of gene products responsible for specific diseases of the nervous system have opened opportunities for novel therapies using gene transfer vectors for gene replacement. Herpes simplex virus (HSV)‐based vectors are particularly well suited for gene delivery to neurons of the central and peripheral nervous systems. The authors have developed methods to delete HSV‐1 IE gene functions and to subsequently introduce foreign genes into the HSV‐1 genome using homologous recombination. This unit describes methods for generating cell lines that complement multiple essential gene deletion mutants as well for generating such replication‐defective virus recombinants and inserting foreign DNA sequences into replication‐defective viral genomes, the last step in preparing a vector. Three support protocols describe methods for preparing virus stocks, titering virus, and preparing viral DNA.
Ex Vivo and In Vivo Gene Delivery to the Brain
Authors: Suhonen Jaana , Ray Jasodhara , Blömer Ulrike , Gage Fred H.
Published: June 2004

This unit describes methods for grafting genetically modified cells for ex vivo delivery of specific genes into the rat brain and direct delivery of transgenes to brain cells in vivo using recombinant viral vectors. These methods assess the function of a gene in the brain. The ex vivo approach of gene transfer to the nervous system depends on genetic manipulation of cells in vitro prior to grafting of the cells into the brain to enable production of a transgene at physiologically significant levels for a long period of time. This unit also includes procedures for transcardial perfusion to fix the tissue prior to analysis of expression and for sectioning of brains by use of a freezing sledge microtome. Thionine staining of tissue sections is also described.
Gene Delivery to Muscle
Authors: Springer Matthew L. , Rando Thomas A. , Blau Helen M.
Published: June 2004

The delivery of genes to skeletal muscle by myoblast implantation, DNA injection, or viral transduction has therapeutic applications for human neuromuscular and systemic disorders, many of which are now represented by transgenic or “knockout” mouse models. This unit describes the isolation and retroviral transduction of mouse myoblasts, the injection of myoblasts and plasmid DNA into mouse muscle, and histological methods for analyzing the recipient muscle. A procedure describing the injection of plasmid DNA into muscle with or without electric charge is also included.
Cancer Vaccines
Authors: Greten Tim F. , Jaffee Elizabeth M.
Published: June 2004

This unit describes the use of retroviral vectors that can be successfully employed for gene transfer into both primary tumor cultures and established cell lines. The unit includes procedures for assaying the stability of the vaccine following gene transfer. Techniques for maintaining the retroviral producer lines and titering the retroviral vectors are also described. A protocol for frozen storage of the vaccine (transduced tumor cells) is provided. In addition, methods are described for characterizing the vaccine cells following gene transfer. Directions for testing the expression of the transferred gene in the transfected tumor line are also given. The final protocol provides suggestions for designing in vivo animal experiments and discusses what has to be observed in a clinical setting.
Isolation of DNA from Forensic Evidence
Authors: Bing David H. , Bieber Frederick R. , Holland Mitchell M. , Huffine Edwin F.
Published: June 2004

This unit covers the many and varied methods for extracting DNA from such diverse specimens as blood, tissue, stamps and envelopes, and cigarette butts, among others. Modifications to the methods that allow the DNA to be used for either PCR or Southern blotbased analyses are also included.
Use of Mouse Models for the Analysis of Human Disease
Authors: Semsarian Christopher
Published: June 2004

The first half of this unit discusses the two main approaches to developing genetically engineered mouse models of human disease (i.e., transgenesis and gene targeting). The experimental steps for each method are discussed. Potential pitfalls, advantages, and limitations are also addressed. The second half of the unit describes two mouse models of hypertrophic cardiomyopathy, which illustrate the value of studying such models of human disease.The first half of this unit discusses the two main approaches to developing genetically engineered mouse models of human disease.
Use of Zebrafish Models for the Analysis of Human Disease
Authors: Zhu Hao , Zon Leonard I.
Published: June 2004

The zebrafish has emerged as a powerful animal model for human diseases. While it has long informed us about the biology of early development, it has recently come into favor for the investigation of clinically relevant problems. Genes conserved from fish to humans can be rapidly analyzed using the zebrafish embryo in what is essentially a transparent in vivo assay. This unit describes methodologies including genetic screening, targeted knockdowns, ectopic overexpression, and transgenesis.
ENU Mutagenesis in the Mouse
Authors: McDonald J. David , Beier David
Published: June 2004

This unit describes the treatment of laboratory mice with the mutagen N‐ethyl‐N‐nitrosourea (ENU) to achieve very highly induced rates of mutation throughout the genome. Further, it describes several popular mating schemes designed to produce animals displaying phenotypes associated with the induced mutations.
Sample Preparation
Authors: McKernan Kevin J.
Published: June 2004

Today's human genetic studies are requiring an ever‐increasing number of sample manipulations. Although a significant investment of time and money are required, automation can considerably reduce human error and improve sample tracking fidelity when applied to such repetitive projects. This unit presents issues involved in the automation of sample preparation for genomic DNA isolation and PCR.
Enzymatic Labeling of DNA
Authors: Tabor Stanley , Struhl Kevin
Published: June 2004

This appendix describes the preparation of uniformly labeled radioactive probes by nick translation and random oligonucleotide‐primed synthesis, as well as the end‐labeling of oligonucleotides by T4 polynucleotide kinase. Accompanying these protocols are methods for removing unincorporated dNTP precursors with spin columns and for measuring the specific activity of a probe by acid precipitation.
Techniques for Mammalian Cell Tissue Culture
Authors: Phelan Mary C.
Published: June 2004

The first section of this appendix discusses basic principles of sterile technique, while the second section discusses medium preparation. The basic protocol describes subculturing of a monolayer culture grown in petri plates or flasks. Support protocols describe freezing of monolayer cells, thawing and recovery of cells, counting cells using a hemacytometer, and preparing cells for transport. An alternate protocol describes freezing suspension cells.
Preparation of DNA from Fixed, Paraffin‐Embedded Tissue
Authors: Fox Edward A.
Published: June 2004

Purification of DNA from fixed, paraffin‐embedded tissue (PET) requires the removal of paraffin, proteins, and, in some cases, heavy metal ions. The resulting DNA is not generally suitable for Southern blot analysis. However, the polymerase chain reaction (PCR) can be used to amplify short regions of this DNA. The Basic Protocol is rapid and involves only a small number of manipulations, thus minimizing the possibility of contamination. The Alternate Protocol involves additional steps including extractions with xylenes and phenol/chloroform. This procedure is recommended as the primary approach when tissue has not been fixed in an optimal manner or as a secondary approach when the Basic Protocol has failed to produce satisfactory DNA.
Analysis of RNA by Northern Blot Hybridization
Authors: Brown Terry , Mackey Karol
Published: June 2004

Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting); unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Because they are single‐stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. Thedescribes blotting and hybridization of RNA fractionated in an agarose‐formaldehyde gel. Alternate protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot‐blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions; these methods are outlined in a.
Introduction to Basic Mouse Handling Techniques
Authors: Donovan John , Brown Patricia , Reeves J.P. , Reeves P.A.
Published: June 2004

Purification of DNA from fixed, paraffin‐embedded tissue (PET) requires the removal of paraffin, proteins, and, in some cases, heavy metal ions. The resulting DNA is not generally suitable for Southern blot analysis. However, the polymerase chain reaction (PCR) can be used to amplify short regions of this DNA. In this appendix, the basic protocol can be performed rapidly and involves only a small number of manipulations, thus minimizing the possibility of contamination. An alternate protocol involves additional steps including extractions with xylenes and phenol/chloroform. This procedure is recommended as the primary approach when tissue has not been fixed in an optimal manner or as a secondary approach when the basic protocol has failed to produce satisfactory DNA.
Karyotyping
Authors: Schreck Rhona R. , Distèche Christine
Published: June 2004

Relevant portions of the new International System for Human Cytogenetic Nomenclature (ISCN 1995) have been reproduced in this appendix (with permission from Karger, the original publisher). The new rules supersede all previous rules and include guidelines for cancer cytogenetics as well as new recommendations for nomenclature when in situ hybridization techniques are used in the analysis of chromosomes.
Human‐Mouse Comparative Maps
Authors: Seldin Michael F.
Published: June 2004

Homology relationships between human and mouse genomes are very useful for identifying human or mouse homologs of disease traits that have been mapped in the other species. Conservation of genomic organization in human and mouse has long been recognized; however, detailed systematic examination of these relationships on a genome‐wide scale has only recently become possible. This appendix presents tables of comparative genetic maps with homology groups for human and mouse chromosomes, sorted by human position. The map locations of 1416 loci (many of which are genes) have been determined, and at least 181 different conserved linkage groups have been defined. Human loci are arranged from the most telomeric marker on the long (q) arm to the most telomeric marker on the short (p) arm of the human chromosome.
Oxidation of N,N‐Disubstituted Hydroxylamines to Nitrones with Hydrogen Peroxide Catalyzed by Polymer‐Supported Methylrhenium Trioxide Systems
Authors: Saladino Raffaele , Neri Veronica , Cardona Francesca , Goti Andrea
Published: June 2004

Poly(4‐vinylpyridine)/methylrhenium trioxide (MTO) compounds IIII and microencapsulated polystyrene/MTO systems IVV are efficient catalysts for the oxidation of secondary hydroxylamines to the corresponding nitrones with H2O2. Complete conversions of substrates and quantitative yields of products are obtained under environmentally friendly experimental conditions and with the use of simple work‐up procedures. Symmetrically substituted hydroxylamines, and non‐symmetrical 3‐substituted and 2‐substituted hydroxypyrrolidines, precursors of nitrones applied in the synthesis of alkaloids and biologically active congeners, have been considered as substrates. The heterogeneous catalysts are stable under the reaction conditions and can be recovered and recycled for at least five times without any appreciable loss in efficiency.
On the Use of Non‐Symmetrical Mixed PCN and SCN Pincer Palladacycles as Catalyst Precursors for the Heck Reaction
Authors: Consorti Crestina S. , Ebeling Gunter , Flores Fabricio R. , Rominger Frank , Dupont Jairton
Published: June 2004

The mixed pincer palladacycles (Me2NCH2(Cl)CCCH2CH2Y‐κN,κC,κY)PdCl (1, Y=PPh2; 2, OPPh2) and (t‐BuSCH2CH2CC(Cl)(o‐NC5H4)‐κS,κC,κN)PdCl 3 have been obtained in high yields by chloropalladation of heterosusbstituted alkynes Me2NCH2CCCH2CH2PPh2, Me2NCH2CCCH2CH2OPPh2 and t‐BuSCH2CH2CC(o‐NC5H4), respectively. The molecular structures of 1 and 3 have been ascertained by means of X‐ray diffraction analysis. The catalytic properties of these mixed donor group pincer‐type palladacycles have been evaluated in the arylation of olefins (Heck reaction). The pincer palladacycle 1 is highly active for the coupling of aryl iodides and aryl bromides with n‐butyl acrylate. In contrast it is only moderately active for the coupling of aryl chlorides substituted with electron‐withdrawing groups and inactive for the coupling of electron neutral and electron deactivated aryl chlorides.
Antimony(V) Chloride‐Benzyltriethylammonium Chloride Complex as an Efficient Catalyst for Friedel−Crafts Acylation Reactions
Authors: Huang An‐ping , Liu Xue‐yuan , Li Lian‐hua , Wu Xiao‐li , Liu Wei‐min , Liang Yong‐min
Published: June 2004

A novel catalytic system, the complex of antimony(V) chloride (SbCl5) and benzyltriethylammonium chloride (TEBA), C6H5CH2NEt3(SbCl5)2Cl complex, is described for Friedel–Crafts acylation reactions of aromatics with acyl and sulfonyl chlorides. The catalyst has a number of useful characteristics, such as ready access, minimal toxicity, reusability, insensitivity to atmosphere and moisture, rapid acylation with high yield, and ease of operation.
The First Highly Enantioselective Alkynylation of Chloral: A Practical and Efficient Pathway to Chiral Trichloromethyl Propargyl Alcohols
Authors: Jiang Biao , Si Yu‐Gui
Published: June 2004

A new, inexpensive chiral amino alcohol‐based ligand, (1S,2S)‐2‐N,N‐dimethylamino‐1‐(4‐nitrophenyl)‐3‐(tert‐butyloxy)propan‐1‐ol, was developed for the asymmetric alkynylation of chloral in high yield with up to 98% ee. The resulting chiral adduct (S)‐1‐trichloromethyl‐3‐phenyl‐2‐propyn‐1‐ol was hydrogenated over 10% Pd/C to give the useful intermediate chiral trichloromethyl carbinol in quantitative yield, which was efficiently transformed into the pharmaceutically important building blocks 2‐hydroxy‐4‐phenylbutanoate and homophenylalanine in high yield with excellent enantiomeric excess.
Palladium/Proazaphosphatrane‐Catalyzed Amination of Aryl Halides Possessing a Phenol, Alcohol, Acetanilide, Amide or an Enolizable Ketone Functional Group: Efficacy of Lithium Bis(trimethylsilyl)amide as the Base
Authors: Urgaonkar Sameer , Verkade John G.
Published: June 2004

A commercially available catalyst system comprising Pd(OAc)2 or Pd2(dba)3 and the proazaphosphatrane ancillary ligand P(i‐BuNCH2CH2)3N (1) for the amination of aryl halides substituted with a phenol, alcohol, acetanilide, amide or ketone group containing an enolizable hydrogen is described. The reaction is performed in the presence of LiN(SiMe3)2 as the base. Other bases tested were either less effective or completely non‐functional.
Highly Efficient Suzuki Cross‐Coupling Catalyzed by Palladium/Phosphine‐Imidazolium Carbene System
Authors: Wang Ai‐E , Zhong Jun , Xie Jian‐Hua , Li Kai , Zhou Qi‐Lin
Published: June 2004

Novel phosphine‐imidazolium salts 2 have been synthesized and successfully used in palladium‐catalyzed Suzuki cross‐coupling. A combination of 0.05 mol % of [Pd(η‐C3H5)Cl]2 and 0.1 mol % of 2b in the presence of 2 equivs. of K3PO4 as base provided coupling products in excellent yields in the reaction of aryl bromides and chlorides with aryl boronic acids.
Iodinated Biaryls Synthesized by the Direct Dehydrodimerization of Iodoarenes Using Phenyliodine(III) Bis(trifluoroacetate) (PIFA)
Authors: Mirk Daniela , Willner Alexander , Fröhlich Roland , Waldvogel Siegfried R.
Published: June 2004

Multiply iodinated biaryls can be prepared in yields up to 75% by direct oxidative coupling reaction of the iodinated arenes. The PIFA‐mediated dehydrodimerization is superior to all other known methods. The developed protocol is reliable and easy to perform.
Geometry effects on ignition in catalytic monoliths
Authors: Ramanathan Karthik , Balakotaiah Vemuri , West David H.
Published: June 2004

The convection–diffusion equations with wall reaction are solved for the case of fully developed laminar flow in monoliths having smooth (circular or flat plate) as well as geometries with sharp corners (triangular, rectangular, square, or sinusoidal). The circumferentially averaged Nusselt/Sherwood numbers along the channel length are calculated and their dependency on the local Damköhler number (ϕ), solid to fluid conductivity ratio (κ = ks/kf), and monolith aspect ratio (dh/L) is studied. As these parameters are varied, the asymptotic Sherwood/Nusselt numbers are bounded by the constant flux (Sh/Nu), the axially constant flux (Sh/Nu), and the constant wall temperature (ShT/NuT) asymptotes. For asymmetric geometries, the difference between Nu and Nu is a measure of the nonuniformity of ignition in the circumferential direction for κ = 0. When wall conduction is significant (κ ≥ 10), ignition can be predicted accurately by using Nu value in a one‐dimensional model, whereas conversion in the mass transfer controlled regime is determined by ShT. © 2004 American Institute of Chemical Engineers AIChE J, 50: 1493–1509, 2004
Effect of temperature and microwave power on the hydrolysis of phenyl acetate
Authors: Carta Renzo , Loddo Laura
Published: June 2004

A study of the influence of microwave radiation, at a frequency of 2.45 GHz on the evolution rate of the hydrolytic decomposition of phenyl acetate evolving in a radiated environment, is presented. The experimental data refer to temperatures of 25, 30, 35, 40, and 45°C at radiating powers of 500 and 1000 mW and to 35°C at the radiating powers of 50, 100, 200, 300, and 400 mW. Experimental data were obtained using the initial rate method and are correlated with a differential procedure. A comparison with the previously obtained results in a nonradiated environment is presented. From this comparison, the catalytic effect of microwave radiation with the reaction evolving in a nonradiated environment is proposed, replacing that running in a solution with catalytic acetate ions. © 2004 American Institute of Chemical Engineers AIChE J, 50: 1523–1529, 2004
Monitoring flames in an industrial boiler using multivariate image analysis
Authors: Yu Honglu , MacGregor John F.
Published: June 2004

An on‐line digital imaging system is developed for monitoring flames in an industrial boiler system. The information extracted from the RGB flame images is used to predict the performance of the boiler system. A practical method based on multivariate image analysis techniques and partial least squares is developed to efficiently extract information from the rapidly time varying flame images, and to predict boiler performance, NOx and SO2 concentration in the off‐gas, and the energy content of an incoming waste fuel stream. The approach is very general and can be applied to a wide range of combustion processes. © 2004 American Institute of Chemical Engineers AIChE J, 50: 1474–1483, 2004
Diet and nutrition in wild mongoose lemurs (Eulemur mongoz) and their implications for the evolution of female dominance and small group size in lemurs
Authors: Curtis Deborah J.
Published: June 2004

Data collected on the feeding behavior, food intake, and chemical analyses of plant foods were used to document seasonal variation in diet and nutrition in Eulemur mongoz in northwestern Madagascar. E. mongoz conforms to the general Eulemur dietary pattern, with a predominantly frugivorous diet supplemented mainly by leaves, flowers, and nectar. Phytochemical analysis revealed high water contents in all the main plant foods; mature fruit and flowers contained the most water‐soluble carbohydrates; immature leaves were richest in protein and essential amino acids; the limiting amino acids in all plant foods were methionine and cystine; ash (mineral) content was highest in petioles and mature leaves; crude lipid content was highest in seeds; and crude fiber content was indistinguishable between immature and mature fruit and leaves. High‐fiber foods were eaten during both seasons; the wet season diet was dominated by high‐energy foods (mature fruit, nectar, and seeds), while the dry season diet contained foods high in energy (mature fruit and flowers) and high in protein (immature leaves) and minerals (mature leaves and petioles). However, nutrient intake did not vary between seasons, implying that nutrient requirements are met throughout the year. These results suggest we draw more conservative conclusions when interpreting dietary variability in the absence of chemical analysis, and also draw into question the idea that nutritional stress is a factor in the timing of reproduction in lemurs and, by extension, is linked to the prevalence of female dominance and small group size in lemurs. Am J Phys Anthropol, 2003. © 2003 Wiley‐Liss, Inc.
Composition of the founding population of Iceland: Biological distance and morphological variation in early historic Atlantic Europe
Authors: Hallgrímsson Benedikt , Ó Donnabháin Barra , Walters G. Bragi , Cooper David M.L. , Guđbjartsson Daníel , Stefánsson Kari
Published: June 2004

We examined the composition of the founding population of Iceland through the study of morphological traits in skeletons from Iceland, Ireland, Norway, and Greenland. This is the first study to address this issue from the Settlement Period of Iceland and contemporary samples from Ireland. We pose the following questions: 1) Was the founding population of Iceland of mixed or homogeneous origin? 2) Is there evidence for a significant Irish cohort in the founding population, as suggested in medieval Icelandic literature? Analysis of biodistance revealed that both Settlement Age and later samples from Iceland showed a greater degree of phenetic similarity to contemporary Viking Age Norwegians than to samples obtained from early medieval Ireland. Analysis of among‐individual morphological variation showed that the Settlement Age population of Iceland did not exhibit an increase in variation in comparison to other populations in the sample, suggesting a relatively homogenous origin. However, estimation of admixture between the Irish and Norwegian populations indicated that 66% of the Icelandic settlers were of Norwegian origin. Comparison of the Icelandic samples to hybrid samples produced by resampling the Viking Age Norwegian and early medieval Irish samples revealed that the Icelandic samples are much closer to the Norwegian samples than expected, based on a 66:34 mixture of Norwegian and Irish settlers. We conclude that the Settlement Age population of Iceland was predominantly (60–90%) of Norwegian origin. Although this population was relatively homogenous, our results do not preclude significant contributions from Ireland as well as other sources not represented in our analysis. Am J Phys Anthropol, 2003. © 2003 Wiley‐Liss, Inc.
Cover Picture: Al50C120H180: A Pseudofullerene Shell of 60 Carbon Atoms and 60 Methyl Groups Protecting a Cluster Core of 50 Aluminum Atoms (Angew. Chem. Int. Ed. 24/2004)
Authors: Vollet Jean , Hartig Jens R. , Schnöckel Hansgeorg
Published: June 2004

A model Al50 cluster is part of the largest known molecular organometallic compound comprised of metal, carbon, and hydrogen. A central Al8 unit (blue in the picture) is made up of 42 Al atoms (silver), 12 of which carry pentamethylcyclopentadienyl (Cp*) groups. The 60 methyl groups of the Cp* ligands (red pentagons) produce a surface topolgy reminiscent of a football. Further details on this Al50Cp${{{\ast 12\hfill \atop 12\hfill}}}$ cluster can be found in the Communication by H. Schnöckel et al. on page 3186 ff.
Seasonal variations in the physical and chemical characteristics of ponds: implications for biodiversity conservation
Authors: Angélibert S. , Marty P. , Céréghino R. , Giani N.
Published: June 2004

The natural evolution of ponds leads to the filling up of these small ecosystems, and human intervention may sometimes be necessary to ensure their continued nature conservation interest.

In the arid karstic region of the ‘Causses du Quercy’ (France), there are numerous ponds bored into parts of the calcareous rock where water cannot percolate. Without human management they are destined to disappear, although they constitute the only available surface water resource on this limestone plateau.

Little information is available about the physical and chemical changes occurring as ponds go through different successional stages. A comparative study of three ponds at various stages of succession allowed an analysis to be made of the seasonal changes in a number of abiotic variables.

There was evidence that the temporal and spatial variability of abiotic factors (such as temperature, water level and dissolved oxygen concentrations) increased as ponds progressed through successional stages.

This study indicated that ponds varied in terms of plant and animal species richness and rarity, temperature, and oxygen concentration at different successional stages. Each pond supported a distinctive fauna. The results suggest that biotic diversity is, in part, driven by local‐scale physical and chemical heterogeneity. Management plans should be drawn at a scale that maintains the heterogeneity of such pond networks.

Copyright © 2004 John Wiley & Sons, Ltd.