Part I Index
David Allison, Bruce Warmack, Mitch Doktycz, Tom Thundat, and Peter Hoyt
Molecular Imaging Group; Health Sciences Research
Division; Oak Ridge National Laboratory; Oak Ridge, TN 37831-6123
Allison: 423/574-6199, Fax: -6210, firstname.lastname@example.org
Warmack: 423/574-6202, Fax: -6210, email@example.com
We have precisely located sequence specific proteins bound to individual DNA molecules by direct AFM imaging. Using a mutant EcoR I endonuclease that site-specifically binds but doesn't cleave DNA, bound enzyme has been imaged and located, with an accuracy of ±1%, on well characterized plasmids and bacteriophage lambda DNA (48 kb). Cosmids have been mapped and, by incorporating methods for anchoring molecules to surfaces and straightening to prevent molecular entanglement, BAC-sized clones could be analyzed.
This direct imaging approach could be rapidly developed to locate other sequence-specific proteins on genomic clones. Enzymatic proteins, involved in identifying and repairing damaged or mutated regions on DNA molecules, could be imaged bound to lesion sites. Transcription factor proteins that identify gene-start regions and other regulatory proteins that modulate the expression of genes by binding to specific control sequences on DNA molecules could be precisely located on intact cloned DNAs.
Conventional gel-based techniques for identifying site-specific protein binding sites must rely upon fragment analysis for identifying restriction enzyme sites, or, for non-cutting proteins, upon gel-shift methods that can only address small DNA fragments. Conversely, AFM imaging is a general approach that is applicable to the analysis of all site-specific DNA protein interactions on large-insert clones. This technique could be developed for high-throughput analysis, can be accomplished by technicians, uses readily available relatively inexpensive instrumentation, and should be a technology fully transferable to most laboratories.
DOE Contract No. DE-AC05-840R21400.
*Improved Cell Electrotransformation by Macromolecules
Our work for 1996 and 1997 will include the following:
2. Study of the efficiency of BAC cloning in DH10B/r cells using new electrotransformation method. Optimization of the procedure for DH10B/r cells.
3. Modernization of the electronic equipment in accordance with results of the biological experiments. To expand the studies, we need to extend the capability of the instrumentation to increase its flexibility and to improve the accuracy and reproducibility of the electric fields we generate by incorporating electronic components with higher tolerances.
DOE Grant No. OR0003393CIS015.
Overcoming Genome Mapping Bottlenecks
Charles R. Cantor
Center for Advanced Biotechnology; Boston University; Boston MA 02215
617/353-8500, Fax: -8501, firstname.lastname@example.org
Most traditional DNA analysis is done based on fractionation of DNA by length. We have, instead, begun to explore the use of DNA sequences as capture and detection methods to expedite a number of procedures in genome analysis.
Triplet repeats like (GGC)n are an important class of human genetic markers, and they are also responsible for a number of inherited diseases involving the central nervous system. For both of these reasons it would be very useful to have a way to monitor the status of large numbers of triplet repeats simultaneously. We are developing methods to isolate and profile classes of such repeats.
In one method, genomic DNA is cut with one or more restriction nucleases,
and splints are ligated onto the ends of the fragments. Then fragments
containing a specific class of repeats are isolated by capture on magnetic
microbeads containing an immobilized simple repeating sequence. The desired
material is then released, and, if necessary, a selective PCR is done to
reduce the complexity of the sample. Otherwise the entire captured sample
is amplified by PCR. The spectrum of repeats is then examined by electrophoresis
on an automated fluorescent gel reader. In our case the Pharmacia ALF is
used, because of its excellent quantitative signal accuracy. A very complex
spectrum of bands is seen representing hundreds of DNA fragments. We have
shown that this spectrum is dramatically different with DNAs from unrelated
individuals, and the spectrum is markedly dependent on the choice of restriction
enzyme, as expected. Repeated measurements on the same sample are highly
reproducible. The ability of the method to detect a specific altered repeat
length in a complex DNA sample has been validated by examining several
individuals with normal or expanded repeat sequences in the Huntington's
disease gene. One very powerful application of this method may be the analysis
of potential DNA differences in monozygotic twins discordant for a genetic
disease. This method can be used to capture genome subsets containing any
interspersed repeat. It will also detect insertions and deletions nearby
such repeats. Methylation differences between sensitive methylation samples
are also detectable when restriction fragments are used.
As an alternative approach, we are developing chipbased methods that can detect the length of a tandemlyrepeating sequence without any need for gel electrophoresis. Here the goal is to build an array of all possible repeat sequence lengths flanked by singlecopy DNA. When an actual sample is hybridized to such an array, the specific alleles in the sample will produce perfect duplexes at their corresponding points in the array and at mismatched duplexes elsewhere. Thus, the task of scoring the repeat lengths is reduced to the task of distinguishing perfect and imperfect duplexes. Currently we are exploring a number of different enzymatic protocols that offer the promise of making such distinctions reliably.
In other work we are using enzyme-enhanced sequencing by hybridization (SBH) as a device for the rapid preparation of DNA samples for mass spectrometry. For example, partially duplex DNA probes can capture and generate sequence ladders from any arbitrary DNA sequence. Current MALDI protocols allow sequence to be read to lengths of 50 to 60 bases. While this is probably insufficient for most de novo DNA sequencing, it is an extremely promising approach for comparative or diagnostic DNA sequencing.
DOE Grant No. DE-FG02-93ER61609.
Preparation of PAC Libraries
Joe Catanese, Baohui Zhao, Eirik Frengen, Chenyan Wu, Xiaoping Guan, Chira Chen, Eugenia Pietrzak, Panayotis A. Ioannou,1 Julie Korenberg,2 Joel Jessee,3 and Pieter J. deJong
Department of Human Genetics; Roswell Park Cancer
Institute; Buffalo, NY 14263
de Jong: 716/845-3168, Fax: -8849
1The Cyprus Institute of Neurology and Genetics; Nicosia, Cyprus
2Cedars Sinai Medical Center; Los Angeles, CA 90048
3Life Technologies, Gaithersburg, MD 20898
Recently, we have developed procedures for the cloning of large DNA fragments using a bacteriophage P1 derived vector, pCYPAC1 (loannou et al. (1994), Nature Genetics 6: 8489). A slightly modified vector (pCYPAC2) has now been used to create a 15-fold redundant PAC library of the human genome, arrayed in more than 1,000 384-well dishes. DNA was obtained from blood lymphocytes from a male donor. The library was prepared in four distinct sections designated as RPCI1, RPCI3, RPCI4 and RPCI5, respectively, each having 120 kbp average inserts. The RPCI1 segment of the library (3X; 120,000 clones, including 25% nonrecombinant) has been distributed to over 40 genome centers worldwide and has been used in many physical mapping studies, positional cloning efforts and in various largescale DNA sequencing enterprises. Screening of the RPCI1 library by numerous markers results in an average of 3 positive PACs per autosome-derived probe or STS marker. In situ hybridization results with 250 PAC clones indicate that chimerism is low or nonexisting. Distribution of RPCI3 (3X, 78,000 clones, less than 1% nonrecombinants, 4% empty wells) is now underway and the further RPCI4 and 5 segments (< 5% empty wells) will be distributed upon request. To facilitate screening of the PAC library, we have provided the RPCI1 PAC library to several screening companies and noncommercial resource centers. In addition, we are now distributing high-density colony membranes at cost-recovery price, mainly to groups having a copy of the PAC library. The combined RPCI1 and 3 segments (6X) can be represented on 11 colony filters of 22x22 cm, using duplicate colonies for each clone. We are currently generating a similar PAC library from the 129 mouse strain.
To facilitate the additional use of large-insert bacterial clones for functional studies, we have prepared new PAC & BAC vectors with a dominant selectable marker gene (the blasticidin gene under control of the betaactin promoter), an EBV replicon and an "update feature". This feature utilizes the specificity of Transposon Tn7 for the Tn7att sequence (in the new PAC and BAC vectors) to transpose marker genes, other replicons and other sequences into PACs or BACs. Hence, it facilitates retrofitting existing PAC/BAC clones (made with the new vectors) with desirable sequences without affecting the inserts. The new vector(s) are being applied to generate second generation libraries for human (female donor), mouse and rat.
DOE Grant No. DEFG0294ER61883 and NIH Grant No. 1R01RG01165.
Development of Affinity Technology for Isolating Individual Human Chromosomes by Third-Strand Binding
Jacques R. Fresco and Marion D. Johnson III
Department of Molecular Biology; Princeton University; Princeton, NJ 08544-1011
609/258-3927, Fax: -6730
Prior to the onset of this grant, solution conditions had been developed for binding a 17-residue third strand oligodeoxyribonucleotide probe to a specific human chromosome (HC) 17 multicopy alpha satellite target sequence cloned into DNA vectors of varying size up to 50 kb. Binding was shown to be both highly efficient and specific. Moreover, initial experiments with fluorescent-labeled third strands and human lymphocyte metaphase spreads and interphase nuclei proved similarly successful. During the current research period, the technology for such third strand-based cytogenetic examination, i.e., Triplex In Situ Hybridization or TISH, of such spreads was perfected, so that it is now a highly reproducible method. Comparison of spreads of different individuals by TISH and FISH analysis has provided a new basis for detecting alpha satellite DNA polymorphisms, the basis of which requires further investigation.
This year work also commenced on the development of comparable probes specific for alpha satellite sequences in HC-X, 11, and 16. The work with HC-X has reached the stage where we are ready to test the probe for TISH-based cytogenetic analysis. Solution studies of the interaction of the probes designed for HC-11 and HC-16 alpha satellite targets are following the well-established path we employed for HC-17 and HC-X. With the expectation of success in these cases during the coming year, the way should be clear for the development and application of comparable probes for alpha satellite sequences of any other human chromosomes that may be of interest, and possibly of other eukaryotic species.
Meanwhile, we have begun to turn our attention to two other goals, one being the exploitation of our probes for the isolation of individual human chromosomes by affinity purification, as we originally proposed. The other goal is to exploit our probes as aids in flow sorting human chromosomes, a direction of work we expect to pursue in collaboration with the Los Alamos National Laboratory, just as soon as they indicate a readiness to do so. Finally, we have begun to evaluate the possibility of using third-strand binding fluorescent probes for detection of single copy genes by means of photon counting, a goal which we plan to undertake with our colleague Robert Austin of our Physics Department.
DOE Grant No. DE-FG02-96ER622202.
Chromosome Region-Specific Libraries for Human Genome Analysis
Eleanor Roosevelt Institute for Cancer Research; Denver, CO 80206
303/333-4515, Fax: -8423, email@example.com
The objective of this project is to construct and characterize chromosome region-specific libraries as resources for genome analysis. We have used our chromosome microdissection and MboI linker-adaptor technique (PNAS 88, 1844, 1991) to construct region-specific libraries for human chromosome 2 and other chromosomes. The libraries have been critically evaluated for high quality, including insert size, proportion of unique vs repetitive sequence microclones, percentage of microclones derived from dissected region, etc.
We have constructed and characterized 11 region-specific libraries for the entire human chromosome 2 (the second largest human chromosome with 243 Mb of DNA), including 4 libraries for the short arm and 6 libraries for the long arm, plus a library for the centromere region. The libraries are large, containing hundreds of thousands of microclones in plasmid vector pUC19, with a mean insert size of 200 bp. About 40-60% of the microclones contain unique sequences, and between 70-90% of the microclones were derived from the dissected region. In addition, we have isolated and characterized many unique sequence microclones from each library that can be readily sequenced as STSs, or used in isolating other clones with large inserts (like YAC, BAC, PAC, P1 or cosmid) for contig assembly. These libraries have been used successfully for high resolution physical mapping and for positional cloning of diseaserelated genes assigned to these regions, e.g. the cloning of the gene for hereditary nonpolypsis colorectal cancer (Cell 75, 12-15, 1993).
For each library, we have established a plasmid sublibrary containing at least 20,000 independent microclones. These sublibraries have been deposited to ATCC for permanent maintenance and general distribution. The ATCC Repository numbers for these libraries are: #87188 for 2P1 library region 2p23p25, comprising 25 Mb); #87189 for 2P2 library (2p21p23, 28 Mb); #87103 for 2P3 library (2p14p16, 22 Mb); #87104 for 2P4 library (2p11p13, 28 Mb); #77419 for 2Q1 library (2q35q37, 28 Mb); #87308 for 2Q2 library (2q33q35, 24 Mb); #87309 for 2Q3 library (2q31q32, 26 Mb); #87310 for 2Q4 library (2q23q24, 19 Mb); #87409 for 2Q5 library (2q21q22, 23 Mb); #87410 for 2Q6 lbrary (2q11q14, 31 Mb); and #87411 for 2CEN library (2p11.1q11.1, 4 Mb). Details of these libraries have been described: Hum. Genet. 93, 557, 1994 (for 2P1 library); Cytogenet. Cell Genet. 68, 17, 1995 (for 2P2 library); Somat. Cell Mol. Genet. 20, 353, 1994 (for 2P3 library); Somat. Cell Mol. Genet. 20, 133, 1994 (for 2P4 library); Genomics 14, 769, 1992 (for 2Q1 library; Somat. Cell Mol. Genet. 21, 335, 1995 (for 2Q2, 2Q3 & 2Q4 libraries); Somat. Cell Mol. Genet. 22, 57, 1996 (for 2Q5, 2Q6 & 2CEN libraries).
Region-specific libraries and short insert microclones for chromosome 2 are particularly useful resources for its eventual sequencing because this chromosome is less exploited and detailed mapping information is lacking. We have also constructed 3 regionspecific libraries for the entire chromosome 18 using similar methodologies, including 18P library (18p11.32p11.1, 22 Mb); 18Q1 library (18q11.1q12.3, 25 Mb); and 18Q2 library (18q21.1q23, 34 Mb). Details of these libraries have been described (Somat. Cell Mol. Genet. 22, 191-199, 1996).
DOE Grant No. DE-FG03-94ER61819.
*Identification and Mapping of DNA-Binding Proteins Along Genomic DNA by DNA-Protein Crosslinking
V.L. Karpov, O.V. Preobrazhenskaya, S.V. Belikov, and D.E. Kamashev
Engelhardt Institute of Molecular Biology; Russian
Academy of Sciences; Moscow 17984, Russia
Fax: +7095/1351405, karpov@genomII.eimb.rssi.ru
In 19951996 we continued to map and identify nonhistone proteins binding at loci along the yeast chromosome. Using DNA-protein crosslinking in vivo, we detected two polypeptides that probably correspond to core subunits of yeast RNApolymerase II in the coding region of the transketolase gene (TKL2). Several nonhistone proteins were detected that bind to the upstream region of TKL2 and to an intergenic spacer between calmodulin (CMD1) and mannosyl transferase (ALG1) genes. The apparent molecular weight of these proteins was estimated. We also developed a new method to synthesize strand-specific probes.
Using DNA-protein crosslinking in vitro, we found the amino acid residues of the Lacrepressor that interacts with DNA. Only Lys33 crosslinks with the Lacoperator in the specific complex.
In addition to Lys33, the N-terminal end of the protein also crosslinks in a nonspecific complex. Our results demonstrate that, in the presence of an inducer, the repressor's N-termini crosslink to the operator's outermost nucleotides. We suggest that binding of an inducer changes the orientation of the DNA-binding domain of the Lac repressor to the opposite of that found for the specific complex.
We plan to use a new method to increase resolution and thus identify amino acids and nucleotides that participate in DNA-protein recognition. The mechanisms of transcription regulation of some yeast genes will thus be further elucidated. Our approaches are based on DNA-protein crosslinking. Detailed analysis will be done for specific and nonspecific complexes, in the presence and absence of inducers. This will allow us to make some conclusions about possible conformational rearrangements in DNA-protein complexes during gene activation at the protein's DNA-binding domains.
DOE Grant No. OR00033-93C1S007.
3. Papatsenko D.A., Priporova I.V., Belikov S.V., and Karpov, V.L. Mapping of DNAbinding proteins along yeast genome by UV-induced DNA-protein crosslinking. FEBS Letters, 1996, 381, 103-105.
4. Belikov S.V., Papatsenko D.A., and Karpov V.L. A method to synthesize strandspecific probes. Anal.Biochemistry, 1996, 240,152-154.
A PAC/BAC Data Resource for Sequencing Complex Regions of the Human Genome: A 2-Year Pilot Study
Julie R. Korenberg
Cedars Sinai Medical Center; University of California;
Los Angeles, CA 90048-1869
310/855-7627, Fax: /652-8010
While the complete sequencing the human genome at 99.99% accuracy is an immediate goal of the Human Genome Project, a serious technical deficiency remains the ability to rapidly and efficiently construct sequence ready maps as sequencing templates. This is particularly problematic in regions with unusual genome structure. An understanding of these troublesome regions prior to genomewide sequencing will provide quality assurance as well as reliable sequencing strategies in these regions.
This proposal will generate a "whole genome" data resource to enable
rapid and reliable sequencing of genomic DNA by the definition and characterization
of the more than 52 regions of high homology now known to be distributed
within unrelated genomic regions and cloned in BACs and PACs. To do this,
Data will be shared with large sequencing efforts, deposited in the 4D database, available with annotation on ftp server and through GDB.
2. Generate contigs of BACs and PACs in regions of complex genome organization. Using STS, EST analyses, fingerprinting, BAC/PAC to BAC/PAC Southerns, end sequence walking in 3.520X libraries, and metaphase/interphase FISH, contigs will be seeded in 25 of the regions of known genome complexity, each of which is estimated as 25 Mb. These data will be used to evaluate and provide independent quality assurance of the STS and Radiation hybrid, and genetic maps in these regions. The most significant of these include 1p36/1q; 2p/q; multiple sites; 8p23 and 8 further sites; 9p/q.
3. Define additional regions of complex genomic structure. Library screening using known members of multiple member retrotransposon and other known repeated sequences defined by the ncbi database, followed by FISH analyzes to determine structure and potential large regions of associated homologies.
Collaboration with other genome and sequencing centers will provide quality control in the generation of sequenceready maps for sequencing templates.
We believe that this effort is important since 1) it will provide a critical mapping tool necessary for the generation of sequence ready maps; 2) if initiated now, the problem areas could be delineated before scale ups to full production occur in major genome centers; 3) represents a modest cost such that the cost of these data would comprise only a small fraction of the cost of the entire genome sequence and would vastly decrease the cost of sequencing errors 4) and could be completed in a, short time (2 to 3 years) so as to be of maximum benefit to sequencing centers. The Principal Investigator in this project is ideally suited for this effort because the group has developed the technology and initiated FISH and genome analyses of over 4000 clones.
We believe that this project represents a critical and timely effort to enable rapid and cost effective human genome sequencing.
Subcontract under Glen Evans' DOE Grant No. DEFC0396ER62294.
Mapping and Sequencing of the Human X Chromosome
D.L. Nelson, E.E. Eichler, B.A. Firulli, Y. Gu, J. Wu, E.Brundage, A.C. Chinault, M. Graves, A. Arenson, R.Smith, E.J. Roth, H.Y. Zoghbi, Y. Shen, M.A. Wentland, D.M. Muzny. J. Lu, K Timms, M. Metzger, and R.A.Gibbs
Department of Molecular and Human Genetics and Human Genome Center; Baylor College of Medicine; Houston, TX 77030
713/798-4787, Fax: -6370 or -5386, firstname.lastname@example.org
The human X chromosome is significant from both medical and evolutionary perspectives. It is the location of several hundred genes involved in human genetic disease, and has maintained synteny among mammals; both of these aspects are due to its role in sex determination and the haploid nature of the chromosome in males. We have addressed the mapping of this chromosome through a number of efforts, ranging from longrange YAC-based mapping to genomic sequence determination.
YAC mapping. The YAC-based map of the X is essentially complete. We have constructed a 40 Mb physical map of the Xp22.3Xp21.3 region, spanning an interval from the pseudoautosomal boundary (PABX) to the Duchenne muscular dystrophy gene. This region is highly annotated, with 85 breakpoints defining 53 deletion intervals, 175 STSs (20 of which are highly polymorphic), and 19 genes.
Cosmid binning. The YAC-based physical is being used in a systematic effort to identify and sort cosmids prepared at LLNL from flow sorted X chromosomes into intervals. Gene identification through use of a common database for cDNA pool hybridization data is continuing. Over 50 YACs have been utilized as probes to the gridded cosmic arrays. These have identified over 9000 cosmids from the 24,000 member library. An additional 4000 cosmids have been identified using a variety of probes, with the bulk coming from cDNA pool probes. More recent emphasis has been placed on BAC clones as their identity for sequencing has been established. These have been identified using the usual methods.
Cosmid contig construction. Creation of longrange continuity in cosmids and BACs proceeds from clones identified by the YAC-based binning experiments. Identification of STS carrying clones is carried out by a combined PCR/ hybridization protocol, and adds to the specificity of the overlap data. Cosmids are grown and DNA is prepared by an Autogen robot. DNAs are digested and analyzed by the AB362 GeneScanner for collection of fingerprint data. The use of novel fluorescent dyes (BODIPY) in this application has increased signal strength markedly. End fragment detection is currently carried out with traditional Southern hybridization, however additional dyes will permit detection without hybridization in the GeneScanner protocol. Data are transferred to a Sybase database and analyzed with ODS (J. Arnold, U. Georgia) software for overlap. ODS output is ported to GRAM (LANL) for map construction. A fully automated approach has yet to be achieved, but this goal is increasingly in reach.
Sequencing. An independently funded project awarded to RAG seeks to develop longrange genomic sequence for ~2 Mb of the human X chromosome. In support of this project, cosmids have been constructed and isolated for the 1.6 Mb region between FRAXA and FRAXF in Xq27.3Xq28. To date, the complete sequences of the regions surrounding the FMR1 and IDS genes have been determined (180 and 130 kb, respectively), along with an additional ~700 kb of the interval. This sequence has led to identification of the gene involved in FRAXE mental retardation. Additional sequence in Xq28 has been determined, including that of a cosmid containing the two genes, DXS1357E and a creatine transporter. This sequence has been duplicated to chromosome 16pl 1 in recent evolutionary history. Comparative sequence analysis reveals 94% sequence identity over 25 kb, and the presence of pentameric repeats which are likely to have mediated the duplication event. A number of technical advances in sequencing have been developed, including the use of BODIPY dyes in AB373 sequencing protocols, which has offered enhanced base calling due to reduced mobility shifting, improved single strand template protocols for much reduced cost, and streamlined informatics processes for assembly and annotation.
DOE Grant Nos. DEFG0592ER6l401 and DEFG0394ER61830 and NIH Grant No.
*Sequence-Specific Proteins Binding to the Repetitive Sequences of
High Eukaryotic Genome
Repetitive sequences occupy the most part of the whole eukaryotic genome but up to the last few years there has not been much interest in their role. The situation changed when alphasatellites in human and minor satellites in mouse became candidates for centromere function responsibility. A number of centromerespecific proteins are under investigation but none seems to distinguish centromeric functions of exact sequences among long arrays of tandemly repeated satellites. The proteins associated with that array are poorly known. We are trying to find out what proteins are involved in maintaining the heterochromatin structure of different types of repetitive sequences.
The major proportion of total genomic satellite DNA remains attached to the nuclear matrix (NM) after DNase1 and high salt treatment. We followed this association in various steps during NM preparation by in situ hybridization with the mouse satellite probe. Two mouse species were used M. musculus and M. spretus. Both contain the same repertoire of satellite DNAs but in different amounts. In M. musculus the centromeric heterochromatin contains major satellite (MA) as the principal component. In M. spretus the minor satellite (MI) is predominant. To test DNA-binding activity of the proteins after chromatography of the soluble NM proteins on cationic and anionic ionexchange columns, gel shift assays were performed with cloned dimer of MA and a trimer of MI. To produce antibodies, the DNA-protein complexes obtained from largescale gelshift assays were isolated and injected into a guinea pig.
The gel shift assay with column fractions from M. musculus NM and MA shows a ladder of complexes. The complexes could be competed out with an excess of MA DNA but not with the same amount of E. coli DNA. Antibodies from the immune serum caused a hypershift of the MA/NM protein complexes. Preimmune serum at the same dilution did not alter the mobility of the complexes. A combination of western and Southern blots allows us to conclude that a protein with a molecular weight of about 80 kD and some similarity to the intermediate filaments is responsible for the MA/NM interaction.
Specific DNA-binding activity to the MI has been tested after column fractionation of the M. spretus NM extract. A ladder of complexes can be competed out with an excess of unlabeled MI but not E. coli or MA DNA. MI contains the CENPBbox sequence, which is the binding site for the protein CENPB, one of the centromeric proteins. Fractions from the NM extract with MIspecific binding activity do not contain CENPB, as shown by western blotting with antiCENPB antibodies.
The same kind of work is going on with human analogs of MA and MI sequences, using large clones of satellite and alphasatellite DNA and nuclear matrices.
There are few satellite DNAbinding proteins isolated, none of them directly from the NM. Our longterm aim is to understand the role of these proteins in heterochromatin formation and in heterochromatin association with NM.
Extracts from handisolated nuclear envelopes from frog oocytes were tested for the specific DNAbinding activity to (T2G4)116. A fragment of Tetrahymena telomere from a YAC plasmid was used as a labelled probe in a gelshift assay. The DNA-protein complexes from the assay were cut out and injected into a guinea pig. The antibodies (AB) obtained stained one protein with an m.w. of about 70 kD in the nuclear envelope of the oocyte, nothing in the inner part of the oocyte, and 70 kD and 120 kD in the frog liver nuclei. The immunofluorescent AB stained fine patches on the oocyte nuclear envelope and a number of intranuclei spots in the frog blood cells.
The electronmicroscope immunogold technique showed that the protein is localized in the outer surface of the oocyte nuclear envelope in cuplike structures. DNA-binding activity to the same sequence has been tested and found in the mouse nuclear matrix extracts. The activity could be eluted from the DEAE52 ion exchange column in 0.15 NaCl. The activity could be competed out with the fragment itself but not with E. coli DNA in the same amounts. AB stained a 70-kD protein in active fractions after ion exchange chromatography. In nuclear matrix preparations, the AB recognized a 120-kD protein as well. The AB caused hypershift of the complexes on the gel shift assay. The AB has some affinity to the keratins. In the mouse cell culture 3T3 line the staining is intranuclei, with fine dots forming chains surrounding dark areas, which do not correspond to the nucleoli.
Similar results were observed when a mouse cell line was transformed with headand tailless human keratin constructs (Bader et al., 1991, J Cell Biol 115:1293). These results suggest that the nuclear proteins detected with the AB may be natural analogs of this artificial keratin construct. The pattern of staining did not resemble the picture of telomerespecific staining. Possibly the protein recognized intragenomic (T2G4)2 sequence, which is present in 25% of murine GenBank sequences rather than telomere. We are going to do immunocytochemical investigations of frog and mouse development in order to determine the point when transcription of the 120- kD protein is initiated and the staining becomes intranuclear.
As a continuation of the previous project the multiple alignment of all the Alu sequences from GenBank is going on. We are also trying to obtain antibodies to the main Alubinding proteins to find out how many proteins could be bound to Alu sequence.
DOE Grant No. OR00033-93C1S014.
*Protein-Binding DNA Sequences
O.L. Polanovsky, A.G. Stepchenko, and N.N. Luchina
Engelhardt Institute of Molecular Biology; Russian
Academy of Sciences; Moscow 117984, Russia
Fax: +7095/1351405, email@example.com
POU domain of Oct2 transcription factor binds octamer sequence ATGCAAAT and a number of degenerated sequences. It has been shown that POUs and POUh domains recognize left and right parts of the octsequence, respectively. The recognized sequences are partly overlapped in the native octamer. In the degenerated recognition sites these core sequences may be separated with a spacer up to four nucleotides. The obtained data changed our view on the number and structure of potential targets recognized on DNA by POU proteins.
Protein-DNA binding is realized due to interaction of a conservative amino acid residues with a DNA target. In POU proteins amino acid residues in positions 47 (Val), 50 (Cys) and 51 (Asn) of POUh domain are absolutely conservative. In order to examine a possible role of Val47 we substituted this residue by each of the 19 other amino acid residues and the interaction of the mutant proteins was investigated with homeospecific site and its variants (ATAANNN) and with oct sequence. It was shown that Ile47 mutant retains the affinity and specificity. Val replacement for Ser, Thr or His partially reduce the affinity.
Asn47 mutant sharply relax the specificity of proteinDNA recognition. Mutants at 47 position have much stronger effects on binding to homeospecific sites than to octamer motifs. Our data indicate that there is not a simple monoletter code of protein/DNA recognition. It has been shown that this recognition is determined not only by the nature of the radicals involved in the contact but also by the structure of DNA binding domain as a whole and probably by cooperative interaction of POUs and POUh domains.
Proposals for 1997. The role of Cys50 in POU domain/DNA recognition will be investigated. This residue is absolutely conservative in POU proteins but it is variable in relative homeoproteins. Our preliminary data allow to suppose that residue at position 50 of POU homeodomain have a key role in discrimination between TAAT-like and octamer sequences. The role of the nuleotides flanking DNA target will be investigated.
DOE Grant No. OR00033-93CIS005.
2. Stepchenko A.G., Polanovsky O.L. (1996) Interaction of Oct proteins with DNA. Molecular Biology, V.30, P.296-302.
3. Stepchenko A.G., Luchina N.N., Polanovsky O.L. The role of conservative Val47 for POU homeodomain/DNA recognition. FEBS Letters, in press.
*Development of Intracellular Flow Karyotype Analysis
V.V. Zenin,1 N.D. Aksenov,1 A.N. Shatrova,1 N.V. Klopov,2 L.S. Cram,3 and A.I. Poletaev
Engelhardt Institute of Molecular Biology; Russian
Academy of Sciences; Moscow 117984, Russia
Poletaev: +7-095/135-9824, Fax: -1405
1Institute of Cytology; Russian Academy of Sciences;
St. Petersburg, Russia
2St. Petersburg Institute of Nuclear Physics; Gatchina, Russia
3Los Alamos National Laboratory; Los Alamos, NM 87545
Instrumentation for univariate fluorescent flow analysis of chromosome
sets has been developed for human cells. A new method of cell preparation
and intracellular staining of chromosome with different dyes was developed
A special magnetic mixing/stirring device was constructed to perform cell membrane breaking. It was placed inside the flow chamber of a serial flow cytometer ATC3000 equipped with additional electronic card for timegated data acquisition . The rupturing of prestained mitotic cells is performed by means of a small magnetic rod vibrating in an alternative magnetic field. The efficiency of mitotic cells breaking with electromagnetic cell breaking device was tested using different human cell lines[2,3].
The device works in a stepwise mode: a defined volume of sample is delivered to the breaking chamber for rupturing mitotic cell (cells) for a defined time period, followed by buffer wash to move the released chromosomes from the breaking chamber to the point of the analysis. The information about the chromosomes appearing at the point of analysis is accumulated in list mode files, making it possible to resolve chromosome sets arising from single cells on the basis of time gating. The concentration of cells in the sample must be kept low to ensure that only one cell at a time enters the breaking device.
The developed software classifies chromosome sets according to different criteria: total number of chromosomes, overall DNA content in the set, and the number of chromosomes of certain type [2,3]. In addition it's possible to determine the presence of extra chromosomes or loss of chromosome types. Thus this approach combines the high performance of flow cytometry (quantitation and high throughput) with the advantages of image analysis (cell to cell karyotype analysis and skills of trained cytogeneticist). The data analysis capabilities offer extensive flexibility in determining important features of the karyotypes under study. This development offers the potential to duplicate most of what is determined by clinical cytogeneticists. The results now obtained are in good accordance with goals of the project formulated before .
DOE Grant No. OR00033-93CIS008.
. V.V. Zenin, N.D. Aksenov, A.N. Shatrova, Y.V. Kravatsky, A. Kuznetsova, L.S. Cram , A.I. Poletaev: "Timegated flow analysis of human chromosomes"; DOE Human Genome Program, Contractor-Grantee Workshop IV, November 1317, 1994; Santa Fe, New Mexico, p. 13.
. V.V. Zenin, N.D. Aksenov, A.N. Shatrova, N.V. Klopov , L.S. Cram, A.I. Poletaev: "Cell by cell flow analysis of human chromosome sets"; DOE Human Genome Program, Contractor-Grantee Workshop V, January 28-February 1,1996; Santa Fe, New Mexico, p. 112.
. Andrei I. Poletaev, Sergei I. Stepanov, Valeri V. Zenin, Nikolay Aksenov, Tatijana V. Nasedkina and Yuri V. Kravazky: "Development of Intracellular Flow-Karyotype Analysis"; DOE Human Genome, 1993 Program Report, p.34-35.
Mapping and Sequencing with BACs and Fosmids
Ung-Jin Kim, Hiroaki Shizuya, and Melvin I. Simon
Division of Biology; California Institute of Technology; Pasadena, CA 91125
Kim: 818/395-4901, Fax: /796-7066, firstname.lastname@example.org
Simon: 818/395-3944, Fax: /796-7066
BACs and fosmids are stable, nonchimeric, and highly representative cloning systems. BACs maintain largefragment genomic inserts (100 to 300 kb) that are easily prepared for most types of experiments, including DNA sequencing.
We have improved the methods for generating BACs and developed extensive BAC libraries. We have constructed human BAC libraries with more than 175,000 clones from male fibroblast and sperm, and a mouse BAC library with more than 200,000 clones. We are currently expanding human library with the aim of achieving total 50X coverage human genomic library using sperm samples from anonymous donors.
The BAC libraries provide resources to bridge the gap between genetic-cytogenetic information and detailed physical characteristics of genomic regions that include DNA sequence information. They also provide reliable tools for generating a highresolution, integrated map on which a variety of information and resources are correlated. Using primarily the human BAC library constructed from fibroblasts, we have assembled a physical contig map of chromosome 22 . First, the entire library was screened by most of the known chromosome 22specific markers that include cDNA, anonymous STS markers, FISH-mapped cosmids and fosmids, YAC-Alu PCR products, FISH-mapped BACs, and flowsorted chromosome 22 DNA. The positive clones have been assembled into contigs by means of the STS-contents or other markers assigned to BAC clones. Most of the contigs were confirmed by using a restriction fingerprinting scheme originally developed by Sulston and Coulson, and modified in our laboratory. Currently, the contigs cover over 80% of the chromosome arm. Various physical or genetic landmarks on this chromosome can now be precisely localized simply by assigning them to BACs or contigs on the map. Using BAC end sequence information from each of the chromosome 22specific BACs, it is now possible to close the gaps efficiently by screening deeper BAC libraries with new probes specific to the ends of contigs.
The resulting BAC contig map is now serving as a road map for sequencing the chromosome. Chromosome 22-specific BAC clones have been distributed to our collaborators including The Sanger Center and Dr. Bruce Roe in University of Oklahoma, and many of the clones have already been sequenced. BAC end sequencing scheme will play a crucial role toward the complete sequencing of chromosome 22, and we are currently sequencing the ends of these BACs directly using the miniprepped BAC DNA as templates.
DOE Grant No. DE-FG03-89ER60891.
 Kim et al. (1996) A Bacterial Artificial Chromosome-based framework contig map of human chromosome 22q. Proc. Natl. Acad. Sci. USA v93 (13): pp6297-6301.
 Venter, C., Smith, H.O., and Hood, L. (1996) Nature 381: pp364-366.
Towards a Globally Integrated, Sequence-Ready BAC Map of the Human Genome
Ung-Jin Kim, Hiroaki Shizuya, and Melvin I. Simon
Division of Biology; California Institute of Technology; Pasadena, CA 91125
Kim: 818/395-4901, Fax: /796-7066, email@example.com
Simon: 818/395-3944, Fax: /796-7066,
BAC clones are ideal for genome analysis since they are nonchimeric, stably maintain large fragment genomic inserts (100300 kb), and it is easy to prepare BAC DNA samples for most types of experiments including DNA sequencing. We have improved BAC cloning technique in the past years and constructed >20X human BAC libraries. As BACs are proving to be the most efficient reagents for large scale genomic sequencing, we intend to increase the depth of the library to 50X genomic equivalence. Using the ESTs, especially the Unigenes that have been chromosomally assigned by other means such as Radiation Hybrid mapping and YAC-based STS content mapping, we plan to organize the BAC library into a mapped resource. The resulting BACEST framework map will provide a high resolution EST (or gene) map and instant entry points for gene finding and large scale genomic sequencing. We also intend to determine the end sequences of the BAC inserts from a significant number of the clones (at least 350,000 clones or 15X genomic equivalence) within two years . All the BACEST mapping data and BAC end sequences will be made available via public databases and WEB servers. The mapping data and end sequence information will dramatically facilitate the process of finding clones that extend the sequenced regions with minimal overlaps. Thus, the tagged BAC libraries will serve as a reliable and facile sequenceready resource and an organizing tool to support and coordinate simultaneously multiple sequencing projects all over the genome.
DOE Grant No. DE-FC03-96ER62242.
 Kim, U.J., Birren, B.W., YuLing Sheng, Tatiana Slepak, Valena Mancino, Cecilie Boysen, HyungLyun Kang, Melvin I. Simon, and Hiroaki Shizuya. (1996) Genomics 34, 213-218.
 Venter, C, Smith, H.O., and Hood, L. (1996) Nature 381: pp364-366.
Generation of Normalized and Subtracted cDNA Libraries to Facilitate Gene Discovery
MarceloBento Soares, MariadeFatima Bonaldo, Pierre Jelenc, and Susan Baumes
Department of Psychiatry; Columbia University; and The New York State Psychiatric Institute; New York, NY 10032
212/960-2313, Fax: /781-3577, firstname.lastname@example.org
Large-scale singlepass sequencing of cDNA clones randomly picked from libraries has proven quite powerful to identify genes and the use of normalized libraries in which the frequency of all cDNAs is within a narrow range has been shown to expedite the process by minimizing the redundant identification of the most prevalent mRNAs. In an attempt to contribute to the ongoing gene discovery efforts, we have further optimized our original procedure for construction of normalized directionally cloned cDNA libraries and we have successfully applied it to generate a number of human cDNA libraries from a variety of adult and fetal tissues . To date we have constructed libraries from infant brain, fetal brain, adult brain, fetal liverspleen, fullterm and 89 week placentae, adult breast, retina, ovary tumor, melanocytes, parathyroid tumor, senescent fibroblasts, pineal glands, multiple sclerosis plaques, testis, B cells, fetal heart, fetal lung, 89 week fetuses and pregnant uterus. Several additional libraries are currently in preparation. All libraries have been contributed to the IMAGE consortium, and they are being widely used for sequencing and mapping.
However, given the large scale nature of the ongoing sequencing efforts and the fact that a significant fraction of the human genes has been identified already, the discovery of novel cDNAs is becoming increasingly more challenging. In an effort to expedite this process further, in collaboration with Greg Lennon (LLNL) we have developed and applied subtractive hybridization strategies to eliminate pools of sequenced cDNAs from libraries yet to be surveyed. Briefly, singlestranded DNA obtained from pools of arrayed and sequence I.M.A.G.E. clones are used as templates for PCR amplification of cDNA inserts with flanking T7 and T3 primers. PCR amplification products are then used as drivers in hybridizations with normalized libraries in the form of singlestranded circles. The remaining singlestranded circles (subtracted library) are purified by hydroxyapatite chromatography, converted to doublestranded circles and electroporated into bacteria. Preliminary characterization of a subtracted fetal liverspleen library indicates that the procedure is effective to enhance the representation of novel cDNAs.
In an effort to enhance the representation of full-length cDNAs in our libraries, as we strive towards our final objective of generating full-length normalized cDNA libraries, we have adapted our normalization protocol to take advantage of the fact that it is now possible to produce singlestranded circles in vitro by sequentially digesting supercoiled plasmids with Gene II protein and Exonuclease III (Life Technologies). This has proven significant because it circumvents the biases introduced by differential growth of clones containing small and large cDNA inserts when singlestrands are produced in vivo upon superinfection with a helper phage.
DOE Grant No. DEFG0291ER61233.
 Bonaldo, M.F., Lennon, G. and Soares, M.B. (1996). Normalization and subtraction: Two approaches to facilitate gene discovery. Genome Research 6, 791-806.
Mapping in Man-Mouse Homology Regions
Lisa Stubbs, Johannah Doyle, Ethan Carver, Mark Shannon, Joomyeong Kim, Linda Ashworth,1 and Elbert Branscomb1
Biology Division; Oak Ridge National Laboratory; Oak Ridge, TN 37831
423/574-0854, Fax: -1283, email@example.com or firstname.lastname@example.org
1Human Genome Center; Lawrence Livermore National Laboratory; Livermore, CA 94550
Numerous studies have confirmed the notion that mouse and human chromosomes resemble each other closely within blocks of syntenic homology that vary widely in size, containing from just a few to several hundred related genes. Within the best-mapped of these homologous regions, the presence and location of specific genes can be accurately predicted in one species, based upon the mapping results obtained in the other. In addition, information regarding gene function derived from the analysis of human hereditary traits or mapped murine mutations, can also be extrapolated from one species to another. However, syntenic relationships are still not established for many human regions, and local rearrangements including apparent deletions, inversions, insertions, and transposition events, complicate most of the syntenically homologous regions that appear simple on the gross genetic level. Because of these complications, the power of prediction afforded in any homology region increases tremendously with the level of resolution and degree of internal consistency associated with a particular set of comparative mapping data. Our groups have been interested in further defining the borders of syntenic linkage groups in human and mouse, upon elucidating mechanisms behind evolutionary rearrangements that distinguish chromosomes of mammalian species, and upon devising means of exploiting the relationships between the two genomes for the discovery and analysis of new genes and other functional units in mouse and man.
One of the larger contiguous blocks of mouse-human genomic homology includes the proximal portion of mouse chromosome 7 (Mmu7). Detailed analysis of this large region of mouse-human homology have served as the initial focus of these collaborative studies. Our results have shown that gene content, order and spacing are remarkably well-conserved throughout the length of this approximately 23 cM/29 Mb region of mouse-human homology, except for six internal rearrangements of gene sequence in mouse relative to man. One of these differences involve a small segment of H19ql3.4 genes whose murine counterparts have been transposed out of the large Mmu7/H19q conserved synteny region into a separate linkage group located on mouse chromosome 17. The six internal rearrangements, including two transpositions and four local inversions, are clustered together at two sites; our data suggest that the rearrangements occurred in a coincident fashion, or were commonly associated with unstable DNA sequences at those sites. Interestingly, both rearranged regions are occupied by large tandemly clustered gene families, suggesting that these locally repeated sequences may have contributed to their evolutionary instability. The structure and conserved functions of genes within these and other clustered gene families located on H19 also represent an active line of interest to our group. More recently, we have extended mapping studies to include clustered gene families located in other chromosomal regions, and are working to define the borders of mouse-human syntenic segments on a broader, genome-wide scale.
DOE Contract No. DE-AC05-96OR22464 and Contract No. W-7405-ENG-48 with Lawrence Livermore National Laboratory.
Positional Cloning of Murine Genes
Lisa Stubbs, Cymbeline Culiat, Ethan Carver, Johannah Doyle, Laura Chittenden, Mitchell Walkowicz, Nestor Cacheiro, Greg Lennon,1 Gary Wright,2 Joe Rutledge,3 Robert Nicholls,4 and Walderico Generoso
Biology Division; Oak Ridge National Laboratory; Oak Ridge, TN 37831-8077
423/574-0854, Fax: -1283, email@example.com or firstname.lastname@example.org
1Human Genome Center; Lawrence Livermore National Laboratory; Livermore, CA 94550
2University of Texas Southwestern Medical Center at Dallas; Dallas, TX 75235
3Children's Hospital and Medical Center; University of Washington School of Medicine; Seattle, WA 98105
4Department of Genetics; Case Western Reserve University; Cleveland, Ohio
Chromosome rearrangements, notably deletions and translocations, have proved invaluable as tools in the mapping and molecular cloning of a acquired and inherited human diseases. Because balanced translocations are cytologically visible, and generally produce profound disturbances in both gene expression and DNA structure without necessarily disturbing the structure of multiple genes, this type of mutation provides an especially valuable "tag" that greatly simplifies mapping, cloning, and assessment of candidate genes associated with a disease. Although balanced translocations are relatively rare in human populations, they are readily induced in the mouse. Using various mutagenesis protocols, we have generated numerous translocation-bearing mutant mouse strains that display an impressive variety of health-related anomalies, including obesity, polycystic kidneys, gastrointestinal disorders, limb and skeletal deformities, neural tube defects, ataxias, tremors, hereditary deafness and blindness, reproductive dysfunction, and complex behavioral defects. The ability to map the genes associated with translocation breakpoints cytogenetically, first crudely through straightforward banding techniques and then to a higher level of resolution using fluorescence in situ hybridization methods, allows us to avoid the costly and time-consuming crosses that are required for the mapping of most mutant genes. With this rapidly-obtained, crude-level mapping information available, we can readily assess possible relationships between newly arising mutant phenotypes and linked candidate genes or related diseases that map to homologous regions of the human genome. Using this approach, we have recently begun to define the map positions of several mutations. Mapping results have led us to the identification of candidate genes for two mutations: one associated with congenital deafness and predisposition to severe gastric ulcers, and another associated with late-onset obesity. So far, we have characterized only a fraction of the mouse strains that comprise this valuable, recently-generated mutant collection in detail. As a integral part of this program, we are actively exploring new strategies and integrating information, technology and resources derived from the Human Genome research effort, that promise to increase the efficiency of breakpoint mapping and cloning dramatically. The mutations are scattered widely throughout the mouse genome corresponding to a broad selection of human homology regions. As new breakpoints are mapped, and large numbers of newly-sequenced cDNA clones are assigned to the mouse and human maps, the potential for rapid association between cloned gene and mapped mutation will increase dramatically. This large collection of murine translocation mutants therefore represents a powerful resource for linking mapped cDNA clones to health-related phenotypes throughout the genome.
In addition to the analysis of translocation mutants, we have also characterized other types of mouse mutations, including: (1) tottering and leaner, allelic mutations associated with ataxia and epilepsy in mice, and representing murine models for human diseases, familial hemiplaegic migraine and episodic ataxia, respectively; and (2) jdf2, a locus associated with mutations causing runting, neuromuscular tremors and male sterility which is located in a mouse region related to the Prader Willi-Angleman syndrome gene interval of human 15q11-q13. Both sets of mutations affect large, complex, and highly conserved genes, and provide important animal models for the exploration of the diverse roles their human counterparts may play in human disease. In concert with these gene cloning studies, we have been involved in exploring new means of exploiting mouse-human genomic conservation in the isolation of functionally-significant sequences from large cloned regions of human DNA. The methods we have developed hold great promise as an efficient tool for gene discovery in cloned genomic regions.
DOE Contract No. DE-AC05-96OR22464.
Human Artificial Episomal Chromosomes (HAECS) for Building Large
Of some 100,000 human genes, only a few thousand have been cloned, mapped or sequenced so far. Much less is known about other chromosomal regions such as those involved in DNA replication, chromatin packaging, and chromosome segregation. Construction of detailed physical maps is only the first step in localizing, identifying and determining the function of genetic units in human cells. Studying human gene function and regulation of other critical genomic regions that span hundreds of kilobase pairs of DNA requires the ability to clone an entire functional unit as a single DNA fragment and transfer it stably into human cells.
We have developed a human artificial episomal chromosome (HAEC) system based on latent replication origin of the large herpes EpsteinBarr virus (EBV) for the propagation and stable maintenance of DNA as circular minichromosomes in human cells.[1,2] Individual HAECS carried human genomic inserts ranging from 60 to 330 kb and appeared genetically stable. An HAEC library of 1500 independent clones carrying random human genomic fragments with average sizes of 150 to 200 kb was established and allowed recovery of the HAEC DNA. This autologous HAEC system with human DNA segments directly cloned in human cells provides an important tool for functional study of large mammalian DNA regions and gene therapy.[3,4]
Current efforts are focused on (a) shuttling large BAC/PAC genomic inserts in human and rodent cells and (b) packaging BAC/PAC/HAEC clones as large infectious Herpes Viruses for shuttling genomic inserts between mammalian cells and (c) constructing bacterialbased human and rodent HAEC libraries. (a) We have designed a "popin" vector, which can be inserted into current BACor PACbased clone via sitespecific integration. This "CRELOXP"-mediated system has been used to establish BAC/PAC up to 250 kb in size in human cells as HAECS. (b) We have obtained packaging of 160-180 kb exogenous DNA into infectious virions using the human lymphotropic EpsteinBarr virus. After delivery into human betalymphoblasts cells the HAEC DNA was stably established as 160-180 kb functional autonomously replicating episomes.[5,7] We have also generated a hybrid BAC/HAEC vector, which can shuttle large DNA inserts, i.e., at least up to 260 kb, between bacteria and human cells. Such a system is being used to develop large insert libraries, whose clones can be directly transferred into human or rodent cells for functional analysis. These HAECderived systems will provide useful molecular tools to study large genetic units in humans and rodents, and complement the functional interpretation of current sequencing efforts.
DOE Contract No. DEFG0591ER61135.
 Sun, T.Q. & Vos, J.M.H. Engineering of 100300 kb of DNA as persisting extrachromosomal elements in human cells using the HAEC system in Methods molec. Genet. (ed. Adolph, K.W.) (Academic Press, San Diego, CA, 1995).
 Vos, J.M.H. Herpes viruses as Genetic Vectors in Viruses in Human Gene Therapy (ed. Vos, J.M.H.) 109140 (Carolina Academic Press & Chapman & Hall, Durham N.C., USA & London, UK, 1995).
 Kelleher, Z. & Vos, J.M. LongTerm Episomal Gene Delivery in Human Lymphoid Cells using Human and Avian Adenoviralassisted Transfection. Biotechniques 17, 1110-1117 (1994).
 Banerjee, S., Livanos, E. & Vos, J.M.H. Therapeutic Gene Delivery in Human betalymphocytes with Engineered EpsteinBarr Virus. Nature Medicine 1, 13031308 (1995).
 Sun, T.Q., Livanos, E., & Vos, J.M.H. Engineering a mini-herpesvirus as a general strategy to transduce up to 180 kb of functional self-replicating human mini-chromosomes. Gene Therapy 3, 10811088 (1996).
 Wang, S. & Vos, J.M.H. An HSV/EBV based vector for High Efficient Gene Transfer to Human Cells in vitro/in vivo. J. Virol. 70, 84228430 (1996).
*Cosmid and cDNA Map of a Human Chromosome 13q14 Region Frequently Lost at B Cell Chronic Lymphocytic Leukemia
N.K. Yankovsky, B.I. Kapanadze, A.B. Semov, A.V.Baranova, and G.E. Sulimova
N.I. Vavilov Institute of General Genetics; Moscow 117809, Russia
+7-095/135-5363, Fax: -1289, email@example.com and firstname.lastname@example.org (send to both addresses)
We are mapping a human chromosome 13q14 region frequently lost at human
blood malignancy cold B cell chronic lymphocytic leukemia (BCLL). The final
goal of the project is to find putative oncosupressor gene lost in the
region at BCLL. We have constructed a cosmid contig between D13S1168 and
D13S25 loci in the region. The interval had been shown to be in the center
of the BCLL associated deletions. The contig consists of more than 100
cosmids from LANL human chromosome 13 specific library (LA13NC01). We estimated
the distance between D13S1168 and D13S25 loci as about 540 kb. We are constructing
a transcriptional map of the region. Seven different cDNA clones were found
with two of the cosmid clones. All cosmids corresponding to the minimal
tilling path between D13S1168 and D13S25 are being used as probes for screening
new cDNA clones. I.M.A.G.E. Consortium (LLNL) cDNA clones assigned to 13q14
will be mapped against the cosmid contig. Mapped cDNA clones will be checked
as candidate oncosupressor genes for BCLL.
Note: The proceedings of the 1997 DOE Human Genome Program Contractor-Grantee Workshop VI, which include updated research abstracts, can be found at: