Lineage-specific expansion of KRAB zinc-finger transcription factor genes: implications for the evolution of vertebrate regulatory networks.
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Animals↗
Biomedical subjects
E Branscomb
Publications and source records attributed to E Branscomb.
Human chromosome 19 and related regions in mouse: conservative and lineage-specific evolution.
To illuminate the function and evolutionary history of both genomes, we sequenced mouse DNA related to human chromosome 19. Comparative sequence alignments yielded confirmatory evidence for hypothetical genes and identified exons, regulatory elements, and candidate genes that were missed by other predictive methods. Chromosome-wide comparisons revealed a difference between single-copy HSA19 genes, which are overwhelmingly conserved in mouse, and genes residing in tandem familial clusters, which differ extensively in number, coding capacity, and organization between the two species. Finally, we sequenced breakpoints of all 15 evolutionary rearrangements, providing a view of the forces that drive chromosome evolution in mammals.
Animals↗
Initial sequencing and analysis of the human genome.
The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
Animals↗
A physical map of the human genome.
The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.
Chromosomes, Artificial, Bacterial↗
Tandem zinc-finger gene families in mammals: insights and unanswered questions.
Evidence for the remarkable conservation of mammalian genomes, in both content and organization of resident genes, is rapidly emerging from comparative mapping studies. The frequent occurrence of familial gene clustering, presumably reflecting a history of tandem in situ duplications starting from a single ancestral gene, is also apparent from these analyses. Genes encoding Kruppel-type zinc-finger (ZNF) proteins, including those containing Kruppel-associated box (KRAB) motifs, are particularly prone to such clustered organization. Existing data suggest that genes in KRAB-ZNF gene clusters have diverged in sequence and expression patterns, possibly yielding families of proteins with distinct, yet related, functions. Comparative mapping studies indicate that at least some of the genes within these clusters in mammals were elaborated prior to the divergence of mammalian orders and, subsequently, have been conserved. These data suggest a possible role for these tandem KRAB-ZNF gene families in mammalian evolution.
Amino Acid Sequence↗
The human homolog of a mouse-imprinted gene, Peg3, maps to a zinc finger gene-rich region of human chromosome 19q13.4.
Peg3 (paternally expressed gene 3) is the first imprinted gene detected in the proximal region of mouse chromosome 7. Because imprinting is a trait that is generally conserved among mammals, and imprinted domains generally encompass several adjacent genes, expression patterns and chromosomal environment of the human counterpart of Peg3 are of special interest. In this study we have localized human PEG3 approximately 2 Mb proximal of the telomere of chromosome 19q, within a region known to carry large numbers of tandemly clustered Krüppel-type zinc finger-containing (ZNF) genes. Peg3 also encodes a Krüppel-type ZNF protein but one that is distinguished from other ZNF gene products by the fact that it carries two novel proline-rich motifs. Comparison between mouse Peg3 and partial human PEG3 gene sequences revealed a high level of conservation between the two species, despite the fact that one of the two proline-rich repeats is absent from the human gene. Our data demonstrate that the human gene is expressed at highest levels in ovary and placenta; mouse Peg3, by contrast, is transcribed at highest levels in the adult brain. These comparative mapping, sequencing, and expression data provide the first clues to the potential activities of PEG3, and generate new tools to aid in the analysis of structure and function of a potentially new imprinted domain located in human chromosome 19q13.4 and mouse chromosome 7.
Age Factors↗
Comparative analysis of a conserved zinc finger gene cluster on human chromosome 19q and mouse chromosome 7.
Several lines of evidence now suggest that many of the zinc-finger-containing (ZNF) genes in the human genome are arranged in clusters. However, little is known about the structure or function of the clusters or about their conservation throughout evolution. Here, we report the analysis of a conserved ZNF gene cluster located in human chromosome 19q13.2 and mouse chromosome 7. Our results indicate that the human cluster consists of at least 10 related Kruppel-associated box (KRAB)-containing ZNF genes organized in tandem over a distance of 350-450 kb. Two cDNA clones representing genes in the murine cluster have been studied in detail. The KRAB A domains of these genes are nearly identical and are highly similar to human 19q13.2-derived KRAB sequences, but DNA-binding ZNF domains and other portions of the genes differ considerably. The two murine genes display distinct expression patterns, but are coexpressed in some adult tissues. These studies pave the way for a systematic analysis of the evolution of structure and function of genes within the numerous clustered ZNF families located on human chromosome 19 and elsewhere in the human and mouse genomes.
Amino Acid Sequence↗
An integrated metric physical map of human chromosome 19.
A metric physical map of human chromosome 19 has been generated. The foundation of the map is sets of overlapping cosmids (contigs) generated by automated fingerprinting spanning over 95% of the euchromatin, about 50 megabases (Mb). Distances between selected cosmid clones were estimated using fluorescence in situ hybridization in sperm pronuclei, providing both order and distance between contigs. An average inter-marker separation of 230 kb has been obtained across the non-centromeric portion of the chromosome. Various types of larger insert clones were used to span gaps between contigs. Currently, the map consists of 51 'islands' containing multiple clone types, whose size, order and relative distance are known. Over 450 genes, genetic markers, sequence tagged sites (STSs), anonymous cDNAs, and other markers have been localized. In addition, EcoRI restriction maps have been generated for > 41 Mb (approximately 83%) of the chromosome.
Base Sequence↗
Fluorescence in situ hybridization mapping of human chromosome 19: mapping and verification of cosmid contigs formed by random restriction enzyme fingerprinting.
Automated restriction enzyme fingerprinting of 7900 cosmids from chromosome 19 and calculation of the likelihood of their overlap based on shared fragments have resulted in the assembly of 743 sets of overlapping cosmids (contigs). We have mapped 22% of the formed contigs (n = 165) and all of the contigs with minimal tiling paths exceeding 6 members (n = 50) to chromosomal bands by fluorescence in situ hybridization using DNA from at least one member cosmid. The estimated average size of the formed contigs is 60-70 kb. Thus, members of a correctly formed contig are expected to lie close to each other in metaphase and interphase chromatin. Therefore, we tested the contig assembly process by comparing the band assignment of two or more members selected from each of 97 contigs. Forty-two of these contigs were further characterized for valid assembly by determining the proximity of members in interphase chromatin. Using these tests, we surveyed a total of 431 joins counted along the minimal tiling path (280 in interphase as well as metaphase) and found 6 erroneous joins, one in each of 6 contigs (6% of tested).
Chromosomes, Human, Pair 19↗
Optimizing restriction fragment fingerprinting methods for ordering large genomic libraries.
We present a statistical analysis of the problem of ordering large genomic cloned libraries through overlap detection based on restriction fingerprinting. Such ordering projects involve a large investment of effort involving many repetitious experiments. Our primary purpose here is to provide methods of maximizing the efficiency of such efforts. To this end, we adopt a statistical approach that uses the likelihood ratio as a statistic to detect overlap. The main advantages of this approach are that (1) it allows the relatively straightforward incorporation of the observed statistical properties of the data; (2) it permits the efficiency of a particular experimental method for detecting overlap to be quantitatively defined so that alternative experimental designs may be compared and optimized; and (3) it yields a direct estimate of the probability that any two library members overlap. This estimate is a critical tool for the accurate, automatic assembly of overlapping sets of fragments into islands called "contigs." These contigs must subsequently be connected by other methods to provide an ordered set of overlapping fragments covering the entire genome.
Genomic Library↗
A high-resolution, fluorescence-based, semiautomated method for DNA fingerprinting.
We describe a fluorescence-based method for the automated analysis of DNA fragments on polyacrylamide gels. A single-stranded oligonucleotide primer (18-mer) with a fluorochrome covalently bound to its 5'-end is annealed to a synthetic oligonucleotide to create a double-stranded oligonucleotide linker with a 5'-overhang complementary to a restriction enzyme site. Cosmid or plasmid DNA is digested with the appropriate restriction enzyme and then ligated to the fluorochrome-labeled linker. The labeled restriction fragments are loaded on a denaturing polyacrylamide gel in a commercially available DNA sequencer. As the restriction fragments migrate through the gel, they intersect a laser beam which excites the fluorochrome-labeled fragment. Fluorescence emission data are captured on a computer in real time and analyzed after the completion of electrophoresis. Fragment length is nearly linearly related to migration time. This method offers very near single-base resolution up to 400 bases and the ability to quantitate fragment size up to 2000 bases. The fluorochrome-labeling chemistry relies on straightforward enzymatic reactions and can be performed in a single reaction tube. Because four different fluorochromes can be used, each of 16 lanes on the gel can be used to analyze four different digest reactions, one in each color. One of the fluorochromes can be used to label size standards in each lane, eliminating interlane variability and allowing more precise estimates of fragment size. We apply the method to the analysis of overlapping cosmids.
Animals↗
Constructing chromosome- and region-specific cosmid maps of the human genome.
A chromosome-specific ordered set of cosmids would be a significant contribution toward understanding human chromosome structure and function. We are developing two parallel approaches for creating an ordered cosmid library of human chromosome 19 and other selected subregions of the human genome. The "bottom up" approach is used to establish sets of overlapping cosmids as islands or "contigs" along the chromosome, while the "top down" approach, using pulsed-field gel electrophoresis and yeast cloning, will establish a large-fragment map and close the inevitable gaps remaining from the "bottom up" approach. Source DNA consists of a single homolog of chromosome 19 from a hamster--human hybrid cell and human fragments cloned in yeast artificial chromosomes. We have constructed cosmid libraries in a vector that facilitates cloning small amounts of DNA, allows transcription of the insert termini, and contains unique sites for partial-digest mapping. Computer simulations of cosmid contig building suggest that near-optimal efficiency can be achieved with high-density restriction fragment digest schemes that can detect 20-30% overlap between cosmids. We developed the chemistry and data analysis tools to compare the ordering efficiencies of several cosmid restriction digest fingerprinting strategies. Restriction fragments from a four-cutter digest are labeled with a fluorochrome, separated by polyacrylamide gel electrophoresis, and detected after laser excitation as they traverse a fixed point in the gel. We have also developed the software to rapidly process the output signal to define and analyze the fragment peaks. Up to three cosmids (or three different digests of the same cosmid) plus a size standard are analyzed simultaneously in a single gel lane.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Monoclonal antibodies recognizing single amino acid substitutions in hemoglobin.
Four monoclonal antibodies (mAb) to non-human primate hemoglobin referred to as Cap-4, Cap-5, Rh-2, and Rh-4, and two mAb to human hemoglobin, referred to as H-1 and H-3 were isolated and were partially characterized. Binding studies with these mAb on a panel of hemoglobins and isolated alpha and beta globin chains revealed a unique reactivity pattern for each mAb. Amino acid sequence analysis of the antigens used to generate the binding data suggests that the specific recognition of certain hemoglobin antigens by each mAb is controlled by the presence of a particular amino acid at a specific position within the epitope. The use of synthetic peptides as antigens confirmed this observation for five of the mAb. No synthetic peptides were tested with the sixth mAb, Rh-2. The amino acids required for binding of mAb Cap-4, Cap-5, Rh-4, and Rh-2 to hemoglobin are alanine at beta 5, threonine at beta 13, glutamine at beta 125, and leucine at alpha 68. The non-human primate hemoglobin antibodies require a specific amino acid that is not present in human hemoglobin. The amino acid required for binding of Cap-4, Cap-5, and Rh-4 could arise by a single base change in the beta globin gene, whereas the amino acid required for Rh-2 binding would only occur if two base changes occurred. Thus these mAb are candidate probes for a somatic cell mutation assay on the basis of the detection of peripheral blood red cells that possess single amino acid substituted hemoglobin as a result of single base substitutions in the globin genes of precursor cells.
Amino Acid Sequence↗
The LLNL high-speed sorter: design features, operational characteristics, and biological utility.
This report describes a dual-beam, high-speed sorter (HiSS) with a droplet production rate of 220,000 s-1 now in routine use at the Lawrence Livermore National Laboratory. The system can process and sort objects at rates in excess of 20,000 s-1. We report here on the development of HiSS, describe its operational characteristics, and evaluate its utility for analysis and purification of human chromosomes, human erythrocytes, and living Chinese hamster ovary cells.
Cell Separation↗