Preface

GENOME STRUCTURE - FUNCTION FROM NUCLEI TO CHROMOSOMES AND NUCLEOSOMES

C. NICOLINI

1. Samples preparation

2. Biophysical probes

2.1 GEL ELECTROPHORESIS

2.2 X-RAY SMALL ANGLE SCATTERING

2.3 STM - AFM IMAGING

2.4 CIRCULAR DICHROISM AND CIRCULAR INTENSITY DIFFERENTIAL SCATTERING

2.5 HIGH RESOLUTION FLUORESCENCE IMAGE ANALYSIS BY CCD

2.6 DIFFERENTIAL SCANNING CALORIMETRY

3. Metaphase chromosomes

4. Interphase chromosomes

4.1 NATIVE NUCLEI

4.1.1 Effect of permeabilization and ionic strength

4.1.2 Effect of fixation

4.1.3 Effect of ethidium bromide intercalation

4.2 ISOLATED POLYNUCLEOSOMAL CHROMATIN

4.2.1 Effect of Ethidium Bromide Intercalation

4.3 MONONUCLEOSOMES

5. Alternatives models of overall genome organization in situ

6. Structure-function relationship

6.1 CELL CYCLE ALTERATIONS

6.2 ENZYMATIC MODIFICATIONS

6.3 DNA SUPERCOIL

6.3.1 Linear versus circular phage DNA

7. Conclusions

References

CHROMOSOMAL DNA LOOPS AND DOMAIN ORGANIZATION OF THE EUKARYOTIC GENOME

S. V. RAZIN

1. Periodical attachment of chromosomal DNA to the nuclear matrix organizes this DNA in large closed loops

2. Characterization of specificity of DNA organization into loops

2.1 INTRODUCTION TO THE PROBLEM

2.2 CHARACTERIZATION OF THE SPECIFICITY OF DNA LOOP ORGANIZATION IN HIGH SALT EXTRACTED NUCLEI

2.2.1 Association of transcriptionally active genes with the nuclear matrix

2.2.2 Permanent sites of DNA attachment to the nuclear matrix

2.3 MAPPING OF DNA LOOP ANCHORAGE SITES USING DNA LOOP EXCISION MEDIATED BY HIGH SALT-INSOLUBLE TOPOISOMERASE II OF THE NUCLEAR MATRIX

2.4 ANALYSIS OF THE SPECIFICITY OF DNA ORGANIZATION INTO LOOPS BY HIGH RESOLUTION IN SITU HYBRIDIZATION OF SPECIFIC PROBES WITH NUCLEAR HALOS

2.5 RECONSTITUTION IN VITRO OF THE COMPLEXES OF DNA WITH THE NUCLEAR MATRIX

3. Is there any relationship between the functional organization of the genome and the chromosomal DNA organization into loops?

3.1 REPLICATION ORIGINS ARE LOCATED AT THE BASES OF DNA LOOPS

3.2 DO THE DNA LOOP ANCHORAGE SITES DEFINE THE BORDERS OF TRANSCRIPTION UNITS OR TRANSCRIPTIONALLY ACTIVE GENOMIC REGIONS?

References

INTERRELATIONSHIPS BETWEEN NUCLEAR STRUCTURE AND TRANSCRIPTIONAL CONTROL OF CELL CYCLE AND TISSUE-SPECIFIC GENES

G. S. STEIN

A. J. VAN WIJNEN

J. L. STEIN

J. B. LIAN

M. MONTECINO

1. Introduction: nuclear architecture contributes to transcriptional control

2. Developmental transcriptional control during proliferation and differentiation: regulation of the cell cycle-dependent histone genes and the bone-tissue-specific osteocalcin gene during progressive expression of the osteoblast phenotype

2.1 THE HISTONE GENE PROMOTER IS A MODEL FOR THE INTEGRATION OF REGULATORY SIGNALS MEDIATING CELL CYCLE CONTROL AT THE G1 S PHASE TRANSITION AND PROLIFERATION DIFFERENTIATION INTERRELATIONSHIPS

2.2 TRANSCRIPTIONAL CONTROL OF THE BONE-SPECIFIC OSTEOCALCIN GENE AT THE ONSET OF EXTRACELLULAR MATRIX MINERALIZATION IN POSTPROLIFERATIVE OSTEOBLASTS

2.2.1 Basal/Tissue Specific Factors and Sequences

2.2.2 Multiple Glucocorticoid Responsive Promoter Domains

2.2.3 The VDRE

2.2.4 Other Osteocalcin Gene Promoter Regulatory Sequences

2.2.5 Upstream Regulatory Sequences

2.2.6 Regulatory Implications of Overlapping and Contiguous Regulatory Domains

3. Nuclear structure supports cell cycle stage-specific histone gene transcription in proliferating osteoblasts

3.1 CHROMATIN STRUCTURE AND NUCLEOSOME ORGANIZATION

3.2 THE NUCLEAR MATRIX

4. Nuclear structure supports developmental and steroid hormone responsive osteocalcin gene transcription during osteoblast differentiation

4.1 CHROMATIN STRUCTURE AND NUCLEOSOME ORGANIZATION

4.2 THE NUCLEAR MATRIX

5. Conclusions and prospects

References

TRANSCRIPTIONAL REGULATION IN A CHROMATIN ENVIRONMENT

A. P. WOLFFE

1. Problems for nuclear processes in chromatin

2. Interaction of trans-acting factors with chromatin

2.1 ACCESSIBILITY OF DNA IN CHROMATIN: AN HISTORICAL PERSPECTIVE

2.2 INTERACTION OF SPECIFIC TRANS-ACTING FACTORS WITH NON-SPECIFIC CHROMATIN

2.2.1 Experiments with class III genes

2.2.2 Experiments with class II genes

3. Specific trans-acting factors and specific chromatin

3.1 TRANS-ACTING FACTOR ACCESS TO DNA IN POSITIONED NUCLEOSOMES IN VITRO

3.2 TRANSCRIPTION FACTOR ACCESS TO DNA IN SPECIFIC CHROMATIN STRUCTURES IN VIVO

4. Relationships between transacting factors, DNase I sensitivity, DNase I hypersensitive sites and chromosomal architecture

5. Trans-acting factors and the local organization of chromatin structure

6. Conclusion

References

NUCLEOSOME AND CHROMATIN STRUCTURES AND FUNCTIONS

Sari PENNINGS

E. Morton BRADBURY

1. Histones

1.1 HISTONE MODIFICATIONS

2. Nucleosome structure

3. Chromatin structure

4. DNA packing ratio of active chromatin

5. Factors involved in active chromatin

6. Nucleosome positioning

7. Effects of VLR histones on nucleosome positions

8. Nucleosome mobility

9. Very Lysine Rich histones suppress nucleosome mobility

10. Transcription through nucleosomes

References

CHROMATIN STRUCTURE AND GENE REGULATION BY STEROID HORMONES

Miguel BEATO(1)

Sebastian CHAVEZ(2)

Karin EISFELD(1)

Christian SPANGENBERG(1)

Mathias TRUSS(1)

1. Introduction

2. Steroid hormone receptors are modular ligand activated transcription factors

3. The receptors can interact directly or indirectly with general transcription factors

4. Steroid hormone receptors participate in transcriptional repression

5. Hormone receptors can be activated by signals acting through other transduction pathways

6. The function of the hormone responsive region of the MMTV promoter is modulated by its assembly in nucleosomes

6.1 THE MMTV PROMOTER IN YEAST

6.2 NFI BINDING TO RECONSTITUTED MMTV NUCLEOSOMES

7. Hormone induction involves remodeling of the chromatin structure of the MMTV promoter

7.1 MULTIPROTEIN COMPLEXES INVOLVED IN CHROMATIN REMODELING

7.2 DNASE I HYPERSENSITIVITY OVER THE MMTV NUCLEOSOME

7.3 MODIFICATIONS OF THE CORE HISTONES

7.4 DISSOCIATION OF DIMERS OF HISTONES H2A AND H2B

7.5 ADDITIONAL MECHANISMS

References

THE NUCLEAR PI CYCLE: ITS RELEVANCE TO NUCLEAR STRUCTURE AND FUNCTION

R Stewart GILMOUR(1)

Alberto M MARTELLI(2)

Lucia MANZOLI(3)

Anna M BILLI(4)

Lucio COCCO(4)

1. Discovery of the nuclear PI cycle

2. Nuclear PI cycle responses

3. Downstream effects of the nuclear PI cycle

3.1 DAG AND PROTEIN KINASE C (PKC)

3.2 NUCLEAR CALCIUM

4. Immunocytochemical evidence for the nuclear PI cycle

4.1 NUCLEAR PHOSPOLIPIDS

4.2 PLC ISOFORMS: WHOLE CELL STUDIES

4.3 PLC ISOFORMS: LOCALISATION IN SUBCELLULAR FRACTIONS

5. Concluding remarks

References

HISTONE ACETYLATION A global regulator of chromatin function

Bryan M. TURNER

1. Chromatin is a major determinant of gene expression in eukaryotes

2. Histones are highly conserved but extensively modified

3. Histones undergo cyclical acetylation and deacetylation

4. Individual lysine residues are selectively acetylated

5. Antibodies to acetylated histones are useful experimental tools

6. Patterns of histone acetylation are likely to be determined primarily by the specificities of the acetylating and deacetylating enzymes

7. Histone acetylation is associated with various basic cellular functions

8. Histone acetylation, DNA replication and chromatin assembly

9. Histone acetylation and cell cycle progression

10. Histone acetylation and transcription

10.1 IMMUNOPRECIPITATION

10.2 YEAST MUTANTS

10.3 TRANSCRIPTION FACTOR BINDING TO NUCLEOSOMES IN VITRO

11. Histone acetylation and genomic marking

12. Histone acetylation, nucleosome surface markers and intranuclear signalling

References

THE ROLE OF STRUCTURE IN COMPLEXES BETWEEN THE p53 DNA BINDING DOMAIN AND DNA RESPONSE ELEMENTS

A. K. NAGAICH(1)

P. BALAGURUMOORTHY(3)

W. M. MILLER(1)

E. APPELLA(2)

V. B. ZHURKIN(3)

R. E. HARRINGTON(1)

Synopsis

1. The p53 protein

2. DNA binding properties of p53

3. Evidence that four p53 DNA binding domain peptides bind natural p53 response elements and bend the DNA

3.1 PREPARATION OF THE P53DBD PEPTIDE USED IN THE VARIOUS STUDIES DISCUSSED IN THIS REPORT

3.2 DETERMINATION OF BINDING STOICHIOMETRY

3.3 BINDING AFFINITIES AND COOPERATIVITY USING ANALYTICAL ULTRACENTRIFUGATION

3.4 THE BINDING STOICHIOMETRY OF P53DBD TO WAF1 AND RGC RESPONSE ELEMENTS IS 4:1 AND THE BINDING IS HIGHLY COOPERATIVE

3.5 P53DBD BINDING TO RESPONSE ELEMENTS BENDS THE DNA

4. Chemical probes footprinting of specific protein-DNA contacts in the complexes of p53dbd with waf1 and the crystallographic response elements

4.1 HYDROXYL RADICAL FOOTPRINTING OF P53DBD BOUND TO THE P21 WAF1 CIP1 AND CHO SEQUENCES

4.2 MISSING NUCLEOSIDE EXPERIMENTS

4.3 METHYLATION AND ETHYLATION INTERFERENCE ASSAYS

4.4 GLUTERALDEHYDE CROSSLINKING EXPERIMENTS OF P21 WAF1 CIP1 SEQUENCES WITH P53DBD

4.5 A COMPARISON OF THE FOOTPRINTING RESULTS FOR P53DBD COMPLEXED WITH THE P21 WAF1 CIP1 AND CHO SEQUENCES

5. Stereochemical model for the p53dbd-DNA complex

6. Summary

References

GENETICS OF HUMAN ASTROCYTIC TUMORS

Webster K. CAVENEE

1. Introduction

2. Genetic defects in brain tumors

3. Effects of gene replacement - intrinsic

4. Effects of gene replacement - extrinsic

5. Angiogenesis

6. Conclusions

References

TUMOR PROGRESSION AND METASTASIS

Georgii P. GEORGIEV

Sergei L. KISELEV

Evgenii M. LUKANIDIN

1. "Metastatic genes" and "metastatic proteins"

1.1 TUMOROGENESIS

1.2 TUMOR PROGRESSION

1.3 MAIN STEPS IN TUMOR PROGRESSION AND METASTASIS

1.4 GENES CONTROLLING VASCULARIZATION

1.5 DETACHMENT OF TUMOR CELLS FROM THE ORIGINAL CELL CLUSTER

1.6 INVASIVE GROWTH

1.7 ATTACHMENT TO ENDOTHELIAL CELLS (ARREST IN CAPILLARY BED) AND EXTRAVASATION

1.8 PUTATIVE METASTASIS SUPPRESSOR GENE NM23

1.9 STRATEGY FOR THE SEARCH OF NEW GENES WITH UNKNOWN FUNCTION INVOLVED IN THE CONTROL OF TUMOR METASTASIS

2. The mts1 gene

2.1 ISOLATION OF THE MTS 1 GENE AND ITS PROPERTIES

2.2 THE ROLE IN TUMOR METASTASIS

2.3 A POSSIBLE ROLE OF THE MTS 1 PROTEIN

2.4 CONTROL OF THE MTS 1 GENE EXPRESSION

3. A novel lymphokine, Tag7, putatively playing a role in tumor progression

3.1 DISCOVERY AND GENERAL PROPERTIES

3.2 EXPRESSION OF THE TAG 7 GENE IN NORMAL TISSUES AND TUMORS

3.3 PROPERTIES OF THE TAG 7 PROTEIN

3.4 ACTION OF TAG 7 EXPRESSION ON TUMOR GROWTH

3.5 OTHER GENES DIFFERENTIALLY EXPRESSED IN METASTATIC VMR TUMORS

4. Conclusion

5. References

THE FUTURE OF DNA SEQUENCING: AFTER THE HUMAN GENOME PROJECT

Charles R. CANTOR

Cassandra L. SMITH

Dong JING FU

Natalia E. BROUDE

Ron YAAR

Maryanne MALONEY

Kai TANG

Joel GRABER

Daniel P. LITTLE

Hubert KOESTER

Robert J. COTTER

1. Results of the Human Genome Project

2. Needs for future DNA sequencing

2.1 GENE FINDING

2.2 DIAGNOSTIC DNA SEQUENCING

2.3 EVOLUTIONARY COMPARISONS

2.4 EXPRESSION SCREENING

2.5 ENVIRONMENTAL MONITORING

2.6 DNA AS AN ADDITIVE

3. Techniques and strategies for DNA sequencing

3.1 CONTEMPORARY DNA SEQUENCING TECHNOLOGY

3.2 IMPROVED DE NOVO METHODS

3.2.1 Electrophoresis in Thin Gels or Capillaries

3.2.2 Single Molecule Sequencing

3.3 IMPROVED COMPARATIVE OR DIAGNOSTIC SEQUENCING METHODS

3.3.1 Sequencing by synthesis

3.3.2 Sequencing by Hybridization

3.3.3 Mismatch Scanning

3.3.4 Mass Spectrometry

3.3.5 Combined approaches and indexing

4. High throughput DNA screening methods

References

LARGE-SCALE CHROMATIN STRUCTURE

A. S. BELMONT

1. Introduction

2. Models of mitotic chromosome structure

3. Transitions into and out of mitosis and interphase chromosome structure

4. Alternative approach to analysis of large-scale chromatin organization

5. Preservation of in vivo chromatin packing allows visualization of large-scale chromatin domains

6. Cell cycle modulation in chromonema fiber structure and folding

7. In vivo confirmation of large-scale chromatin fibers

8. Summary and future directions

References

PLANT GENE TECHNOLOGY

I. L. BAGYAN

I. V. GULINA

A. S. KRAEV

V. N. MIRONOV

L. V. PADEGIMAS

M. M. POOGGIN

E. V. REVENKOVA

A. V. SHCHENNIKOVA

O. A. SHOULGA

M. A. SOKOLOVA

J. VICENTECARBAJOSA

G. A. YAKOVLEVA

K. G. SKRYABIN

1. Transformation of Cotton (Gossipium hirsutum I.) with a Supervirulent Strain of Agrobacterium tumefaciens A281 [64]. Construction of a Disarmed Derivative of the Supervirulent Ti Plasmid ptibo542

2. 5'-Regulatory Region of Agrobacterium tumefaciens t-DNA Gene 6b irects Organ-specific, Wound-inducible and Auxin-inducible Expression in Transgenic Tobacco [6].

3. Molecular Characteristics of the Chalcone Synthase Gene Family from Two Cotton Species [13]. Identification of Chalcone Synthase Genes Specifically Expressed in Petals of Two Cotton Species [14].

4. Conserved Structure and Organization of B Hordein Genes in the Hor2 Locus of Barley [83]

5. The Obtaining Transgenic Plants Resistant to Viruses, Herbicides, Insects.

5.1 TRANSGENIC PLANTS RESISTANT TO HERBICIDE PHOSPHINOTHRICIN [59]

5.2 TRANSGENIC POTATO PLANTS RESISTANT TO Y-VIRUS INFECTION [60, 61, 71]

5.3. TRANSGENIC POTATO PLANTS RESISTANT TO COLORADO POTATO BEETTLE [31]

6. Conclusions

7. References

THE 5'-UNTRANSLATED LEADERS OF BSMV RNAgamma AND PVX COAT PROTEIN mRNA AS TRANSLATIONAL ENHANCERS IN TOBACCO PROTOPLASTS

I. L. BAGYAN

A. S. KRAEV

G. E. POZMOGOVA

K. G. SKRYABIN

1. Materials and Methods

2. Results and Discussion

3. Acknowledgments

4. References

Subject Index