A new bacteriophage P1-derived vector for the propagation of large human DNA fragments.

We have designed a P1 vector (pCYPAC-1) for the introduction of recombinant DNA into E. coli using electroporation procedures. The new cloning system, P1-derived artificial chromosomes (PACs), was used to establish an initial 15,000 clone library with an average insert size of 130-150 kilobase pairs (kb). No chimaerism has been observed in 34 clones, by fluorescence in situ hybridization. Similarly, no insert instability has been observed after extended culturing, for 20 clones. We conclude that the PAC cloning system will be useful in the mapping and detailed analysis of complex genomes.

Eleven distinct VH gene families and additional patterns of sequence variation suggest a high degree of immunoglobulin gene complexity in a lower vertebrate, Xenopus laevis.

Lower vertebrate species, including Xenopus laevis, exhibit restricted antibody diversity relative to higher vertebrates. We have analyzed more than 180 VH gene-containing recombinant clones from an unamplified spleen cDNA library by selective sequencing of JH and CH positive clones following iterative hybridization screening with family-specific VH probes, 11 unique families of VH genes, each associated with a unique genomic Southern blot hybridization pattern, are described and compared. Considerable variation in the number of hybridizing components detected by each probe is evident. The nucleotide sequence difference between VH families is as great as, if not more than, that reported in other systems, including representatives of the mammalian, avian, and elasmobranch lineages. Some Xenopus Ig gene families encode alternative amino acids at positions that are otherwise invariant or very rarely substituted in known Igs. Furthermore, variations in complementarity determining region sequences among members of the same gene family and high degrees of DH and JH region complexity are described, suggesting that in at least this lower vertebrate species, the diversity of expressed Ig VH genes is not restricted.

Human Txk: genomic organization, structure and contiguous physical linkage with the Tec gene.

Txk is a Tec-family tyrosine kinase expressed in mouse and human T lymphocytes. Among the Tec kinases, Txk is unique in that its amino terminal region does not include a pleckstrin homology domain or other known extended functional region. Txk is encoded at human chromosome 4p12 and at a recognized region of conserved synteny on mouse chromosome 5. The genomic organization of Txk consists of 15 exons with strong exon-intron organizational homology to Btk, the only other Tec-family kinase for which the genomic structure is fully known. The human Tec gene also maps to 4p12 and, based on limited studies reported here, possesses organizational homology with Btk and Txk. We have sequenced a continuous region of DNA that contains 3' Tec and 5' Txk exons separated by only a approximately 1.5 kb intergenic region containing the putative promoter region of Txk. The close physical linkage of these Tec-family tyrosine kinases, which are expressed in different hematopoetic cell lineages, suggests their potential for coordinate cis-regulation.

Generation of a P1 artificial chromosome library of the Southern pufferfish.

We describe the generation of a P1 artificial chromosome genomic library from the Southern pufferfish, Spheroides nephelus. The arrayed library consists of approximately 30,000 clones and has an average insert size of 125-150 kb. The coverage is estimated to encompass seven to eight genome equivalents. The library has been used for isolating numerous genomic clones and for establishing contigs of several multigene families. Analysis of several of the clones from this library suggests a preponderance of CA repeat tracts relative to their abundance in humans. The library and high-density filters have been made available to the scientific public through genomics distribution companies.

Evolution of chordate hox gene clusters.

In this article, we consider the role of the Hox genes in chordate and vertebrate evolution from the viewpoints of molecular and developmental evolution. Models of Hox cluster duplication are considered with emphasis on a threefold duplication model. We also show that cluster duplication is consistent with a semiconservative model of duplication, where following duplication, one daughter cluster remains unmodified, while the other diverges and assumes a new architecture and presumably new functions. Evidence is reviewed, suggesting that Hox gene enhancers have played an important role in body plan evolution. Finally, we contrast the invertebrates and vertebrates in terms of genome and Hox cluster duplication which are present in the latter, but not the former. We question whether gene duplication has been important in vertebrates for the introduction of novel features such as limbs, a urogenital system, and specialized neuromuscular interactions.

Genome biology of the cyclostomes and insights into the evolutionary biology of vertebrate genomes.

The jawless vertebrates (lamprey and hagfish) are the closest extant outgroups to all jawed vertebrates (gnathostomes) and can therefore provide critical insight into the evolution and basic biology of vertebrate genomes. As such, it is notable that the genomes of lamprey and hagfish possess a capacity for rearrangement that is beyond anything known from the gnathostomes. Like the jawed vertebrates, lamprey and hagfish undergo rearrangement of adaptive immune receptors. However, the receptors and the mechanisms for rearrangement that are utilized by jawless vertebrates clearly evolved independently of the gnathostome system. Unlike the jawed vertebrates, lamprey and hagfish also undergo extensive programmed rearrangements of the genome during embryonic development. By considering these fascinating genome biologies in the context of proposed (albeit contentious) phylogenetic relationships among lamprey, hagfish, and gnathostomes, we can begin to understand the evolutionary history of the vertebrate genome. Specifically, the deep shared ancestry and rapid divergence of lampreys, hagfish and gnathostomes is considered evidence that the two versions of programmed rearrangement present in lamprey and hagfish (embryonic and immune receptor) were present in an ancestral lineage that existed more than 400 million years ago and perhaps included the ancestor of the jawed vertebrates. Validating this premise will require better characterization of the genome sequence and mechanisms of rearrangement in lamprey and hagfish.

Genome size variation in North American minnows (Cyprinidae). II. Variation among 20 species.

Genome sizes (nuclear DNA contents) from 200 individuals representing 20 species of North American cyprinid fishes (minnows) were examined spectrophotometrically. The distributions of DNA values of individuals within populations of the 20 species were essentially continuous and normal; the distribution of DNA values among species was continuous and overlapping. These observations suggest that changes in DNA quantity in cyprinids are small in amount, involve both gains and losses of DNA, and are cumulative and independent in effect. Significant heterogeneity in mean genome size occurs both between individuals within populations of species and among species. The former averages maximally around 6% of the cyprinid genome and is nearly the same as the amount of DNA theoretically needed for the entire cyprinid structural gene component. The majority of the DNA content variation among the 20 species is distributed above the level of individuals within populations. Comparisons of average genome size difference or distance between individuals drawn from different levels of taxonomic organization indicate that considerably greater divergence in genome size has occurred in the extremely speciose cyprinid genus Notropis as compared with other North American cyprinid genera. This may suggest that genome size change is concentrated in speciation episodes. Finally, no associations were found between interspecific variation in genome size and five life-history characters. This suggests that much of the variation in genome size within and among the 20 species may be phenotypically inconsequential.

Chromomycin staining of vertebrate chromosomes: enhancement of banding patterns by NaOH.

A simple technique is described for staining vertebrate chromosomes with the GC-base pair specific DNA fluorochrome, chromomycin A3. Addition of NaOH crystals to a glycerol mounting medium resulted in good differentiation of chromosome bands and eliminated the need for counterstaining.

Complete nucleotide sequence of an immunoglobulin heavy-chain gene and analysis of immunoglobulin gene organization in a primitive teleost species.

The immunoglobulin heavy-chain variable region (VH) locus in a phylogenetically primitive teleost (Elops saurus) has been characterized by a strategy that relied initially on cross-hybridization between genomic VH segments and a murine VH probe. Using a homologous (Elops) VH probe and DNA sequencing, this gene family has been shown to be complex and to contain overt pseudogenes. A homologous probe also has been used to isolate a full copy length cDNA containing constant (CH) as well as joining (JH) and VH regions. Genomic analyses using CH-, JH-, and VH-specific probes have demonstrated the presence of only a single hybridizing CH and several JH elements. JH-CH linkage is less than or equal to 3.6 kilobases (kb) and VH-CH linkage is less than or equal to 100 kb, as estimated by field-inversion gel electrophoresis. An additional VH family sharing less than 50% nucleotide identity with the prototype Elops VH sequence is described. Taken together, these results suggest that the immunoglobulin VH locus in a comparatively primitive teleost resembles the VH locus in mammals, but not that found in the more phylogenetically distant elasmobranchs. The evolutionary radiations of cartilaginous and bony fishes are associated with a dramatic change in the organization and, presumably, regulation of immunoglobulin genes. The origins of the modern VH gene locus can be traced to the primitive teleost fishes.

A nonradioactive method for improved restriction analysis and fingerprinting of large P1 artificial chromosome clones.

The P1 artificial chromosome (PAC) cloning system is very useful for physical mapping, however, the large insert sizes cause difficulty in routine restriction analysis. In order to facilitate restriction mapping and fingerprinting, we have developed a simple, nonradioactive method for end-labeling and detection of restriction fragments from PAC clones. This method is very easy to implement, gives good differentiation of restriction fragments, and uses comparatively small amounts of DNA. We have used this method for restriction analysis of PAC clones containing inserts from human as well as from lower vertebrates. The method should also be applicable to other large-insert plasmid systems.

Generation of a zebrafish P1 artificial chromosome library.

We have constructed a genomic P1 artificial chromosome library from the zebrafish. The library has been arrayed and archived in two hundred seventy-one 384-well microtiter dishes. It encompasses four to five genome equivalents with an average insert size of approximately 115 kb and is readily accessible to the scientific community. The library has been used by numerous investigators in the community and shown to be a useful reagent for chromosomal walking and positional cloning.

Two distinct immunoglobulin heavy chain isotypes in a primitive, cartilaginous fish, Raja erinacea.

Immunoglobulin heavy chain genes in Raja erinacea (little skate) are organized in clusters consisting of VH, DH, JH segments and CH exons (1). An immunoglobulin heavy chain mu-like isotype that exhibits 61-91% nucleotide sequence identity in coding segments to the Heterodontus francisci (horned shark) mu-type immunoglobulin is described. The overall length of the mu-type clusters is approximately 16 kb; transmembrane exons (TM1 and TM2) are located 3 to CH exon 4 (CH4). In three of four TM-containing genomic clones, a significant deletion is present in TM1. A second isotype of Raja immunoglobulin heavy chain genes has been detected by screening a spleen cDNA library with homologous Raja VH- and CH1-specific probes complementing the respective regions of the mu-like isotype. Weak hybridization with VH-specific probes and no discernable hybridization with C mu-specific probes were considered presumptive evidence for a second immunoglobulin isotype that nominally is designated as X-type. The Vx region of the X-type cDNA is approximately 60% identical at the nucleotide (nt) level to other Raja VH segments and thus represents a second VH family. Putative Dx and Jx sequences also have been identified. The constant region of the X-type immunoglobulin heavy chain gene consists of two characteristic immunoglobulin domains and a cysteine-rich carboxy terminal segment that are only partially homologous with the mu-like isotype. Genomic Southern blotting indicates that the V and C segments of both immunoglobulin heavy chain isotypes are encoded by complex multigene families. Vx- and different Cx-specific probes hybridize to different length transcripts in northern blot analyses of Raja spleen RNA suggesting that the regulation of expression of the X-type genes may involve differential RNA processing.

Ikaros expression as a marker for lymphoid progenitors during zebrafish development.

The Ikaros gene encodes a transcription factor that, in mice, has been shown to be essential for the correct differentiation of B and T lymphocytes and is expressed in all cells of the lymphoid lineage, including pluripotent hematopoietic stem cells. During development in zebrafish, Ikaros expression begins in lateral mesoderm, and continues in the intermediate cell mass (ICM), which is derived from lateral mesoderm and has been shown to generate primitive hematopoietic precursors. Cells expressing Ikaros were then seen on the ventral side of the dorsal aorta, known to be a location of definitive hematopoietic precursors in birds and mammals. Ikaros-expressing cells were also found in the pharyngeal region, near the forming thymus. Later, such cells were seen in the pronephros, the site of hematopoiesis in adult fish. The timing of appearance of Ikaros-expressing cells suggests that, similar to other vertebrates, lymphocytes in the thymus arise from hematopoietic tissue located near the dorsal aorta or in the ICM.

The genomic organization of immunoglobulin VH genes in Xenopus laevis shows evidence for interspersion of families.

The complete genomic sequences of Xenopus laevis immunoglobulin heavy chain variable region (VH) genes comprising families IV-XI are reported. Using VH family-specific probes, linkage relationships for Xenopus VHI-VHXI have been determined. With the possible exceptions of VHIII and VHVII, Xenopus VH genes appear to be interspersed. When from two to five VH segments are identified in individual clones, the elements are found to be in the same relative transcriptional orientation. Although the relationships of promoter sequences, including the regulatory octamer, resemble those seen in other vertebrate VH genes, several Xenopus VH families are associated with additional 5' octamer sequences and octamer-like motifs. The similarities between the genomic organization of VH genes in Xenopus and higher vertebrates, indicate an early phylogenetic emergence of the mammalian-type of gene organization and regulation.

Evolution of Hoxa-11 in lineages phylogenetically positioned along the fin-limb transition.

HOXA11 is a transcription factor implicated in paired appendage development. To identify signatures of evolutionary change in the structural, and putative functional, domains of HOXA11, we studied its evolution in tetrapod and nontetrapod lineages that represent approximately 1.5 billion years of evolutionary time. Here, Hoxa-11 gene proper sequences were determined for frog (Xenopus tropicalis), coelacanth (Latimeria chalumnae), common zebrafish (Danio rerio; Hoxa-11a and Hoxa-11b paralogs), and giant zebrafish (D. aequipinnatus; Hoxa-11b) and aligned against previously published Hoxa-11 sequences of human, mouse, chick, and newt. Based on aligned Hoxa-11 amino acid sequences, the protein was demarcated into three segments: Domains I (N-terminal) and III (homeobox + C-terminal), which varied slightly in rates and patterns of evolution, and a variable, overall hydrophilic region (HyD), which partially overlaps with Domain I. As judged by character reconstructions of HOXA11 Domains I and III, no significant changes in rates of coding sequence evolution occurred in tetrapods (frog and chick), relative to coelacanth (a lobe-finned fish), i.e., across the fin-limb transition. Accelerated rates of Hoxa-11 coding sequence evolution were observed for the mammalian and newt lineages. This was shown to be a gene-specific phenomenon. The duplicated Hoxa-11a and Hoxa-11b genes of zebrafish exhibited accelerated rates of evolution and accumulated substitutions at sites that are conserved among coelacanth and all tetrapods examined. Amino acid sequence comparisons of the HyD of HOXA11 suggested that a putative repressor subdomain, containing stretches of consecutive alanine residues, emerged within the tetrapods. A high degree of nucleotide conservation in the 5' half of the Hoxa-11 intron was observed for tetrapod and nontetrapod lineages. Using electrophoretic mobility shift assays, a 35-bp intron sequence, which is 100% conserved in all Hoxa-11 loci except for the zebrafish Hoxa-11a paralog, was found to bind protein(s) in HeLa and chick whole-cell extracts.

Marked improvement of PAC and BAC cloning is achieved using electroelution of pulsed-field gel-separated partial digests of genomic DNA.

We describe a simple electroelution method for purifying large, gel-fractionated DNA molecules that alleviates the need for melting of the agarose and subsequent enzymatic agarose digestion. The method yields DNA that is visibly more intact than that purified from a standard agarose-digestion protocol and is more amenable to large-fragment cloning with PAC and BAC vectors. These findings are notable in that PAC and BAC library construction is a very labor-intensive and costly procedure, such that any net improvement in cloning efficiency is highly advantageous. This method also should prove useful towards other applications which require purification of very large DNA molecules, such as YAC cloning.