Antagonistic effects of FGF4 on BMP induction of apoptosis and chondrogenesis in the chick limb bud.

In an effort to define the roles of bone morphogenic proteins (BMPs) and fibroblast growth factors (FGFs) during chick limb development more closely, we have implanted beads impregnated with these growth factors into chick limb buds between stages 20 and 26. Embryos were sacrificed at the time the bone chondrocyte condensations first appear (stages 27-28). Implantation of beads containing BMPs at the earlier stages (20-22) caused apoptosis to occur, in the most severe cases leading to complete limb degeneration. Application of FGF4, either in the same, or in a different bead, prevented the BMP-induced apoptosis. We argue that the apoptosis observed on removal of the AER prior to stage 23 of development could be brought about by BMPs. The action of epithelial FGF in preventing BMP-mediated apoptosis in the mesenchyme would define a novel aspect of epithelial-mesenchymal interactions. Implanting the BMP4 beads into the core of the limb bud a day later (stages 25-26) caused intense chondrogenesis rather than apoptosis. FGF4 could again nullify this effect and by itself caused a reduction in bone size. This is the reverse of the functional relationship these growth factors have in mouse tooth specification (where it is BMP4 that inhibits the FGF8 function), and suggests that the balance between the effects of FGFs and BMPs could control the size of the chondrocyte precursor cell pool. In this way members of these two growth factor families could control the size of appendages when they are initially formed.

A cluster of transfer RNA genes (TRM1, TRR3, and TRAN) on the short arm of human chromosome 6.

We have isolated two lambda clones that contain three transfer RNA (tRNA) genes (TRM1, TRR3, and TRAN). Both clones map to the same region (6p21.2-p22.3) of the short arm of chromosome 6. One clone contains a methionine tRNA gene and also an arginine tRNA gene, the first such human gene to be described. The other clone contains an alanine tRNA gene, again the first such human gene to be reported, and it differs from the species of human alanine tRNA transcripts sequenced to date. These clones have been used to investigate the structure of this tRNA gene cluster. The results of both conventional and pulsed-field gel analysis suggest that the alanine tRNA gene is a member of a low-copy repeat series at this location. The other clone is not located within this domain and appears to be a unique segment of DNA. Nevertheless, we also show that at least half of the methionine tRNA genes are located on the short arm of this chromosome, and if these are also located at 6p21.2-p22.3, this would constitute another major tRNA locus in human.

A primate transfer RNA gene cluster and the evolution of human chromosome 1.

The localisation of tRNA(Asn) gene clusters in the karyotypes of primates has been studied by means of in situ hybridisation. In the human and orangutan (Pongo pygmaeus) karyotypes there are two such gene clusters, one each on the long and short arms of chromosome 1. Old World monkeys, however, contain both gene clusters on their equivalent of the human chromosome 1 short arm, which can be explained by a pericentric inversion which (amongst other chromosome changes) distinguishes the human and Old World monkey chromosomes 1. The capuchin (Cebus appella), however, a New World monkey, has only one tRNA(Asn) gene cluster, at least on the elements equivalent to human chromosome 1. This cluster is located proximal to the centromere on a chromosome that has been tentatively identified (by others) as the equivalent of the long arm of human chromosome 1. Should this prove to be correct, it would indicate that the large primate metacentric came into being in the form found today in the great apes, rather than in the form currently found in Old World monkeys. These data further show that the tRNA(Asn) gene cluster has been split in two since before the Old World monkeys and hominids diverged, i.e., over 30 million years ago, and also that the original transfer of these genes from one arm of chromosome 1 to the other was unlikely to have involved a pericentric inversion but, rather, some form of replicative transposition.

Genomic organization of the human asparagine transfer RNA genes: localization to the U1 RNA gene and class I pseudogene repeat units.

Previously isolated human DNA clones containing asparagine transfer RNA (tRNAAsn) genes have been used to determine the genomic organization of this multigene family in man. One clone also contained a gene for U1 RNA, and so the organization of the two multigene families could be directly compared. The majority, and perhaps all, of the human tRNAAsn genes map to the same chromosome bands as do the U1 RNA true genes and class I pseudogenes located on the short and long arms, respectively, of chromosome 1. These two gene clusters were independently isolated using a somatic-cell hybrid minipanel, and use of repeat-unit DNA polymorphisms showed that one tRNA gene clone maps to the short-arm gene cluster and the other to the long-arm gene cluster. Electron microscopy of heteroduplexes between these two clones showed duplex formation along the proposed region of overlap between them, indicating that the short- and long-arm gene clusters are structurally related. I suggest that the split into two distinct loci was facilitated by a pericentric chromosome inversion. This would have had the effect of positioning the genes currently on the long arm adjacent to the centromeric heterochromatin, perhaps resulting in a "position effect" on transcription of these genes. Restriction fragments of different sizes were found that were common to a majority of repeat units, depending on the restriction enzyme being used. Pulsed-field electrophoresis revealed that fragments of molecular weight of 180 kb were common to each unit (or multiples of units). These fragments also contained U1 RNA gene sequences. I therefore propose that these two gene families are closely linked on repeat units (or multiples of units) of 180 kb in size, which are probably organized in tandem arrays.

On the mechanism of amplification of satellite II DNA sequences of the domestic goat Capra hircus.

A restriction enzyme analysis of the satellite II DNAs of the domestic goat Capra hircus, sheep Ovis aries and ox Bos taurus (p = 1.720, 1.723 and 1.722 g/cm3, respectively) has been carried out and shows that, although all three are composed of repeat units of 700 base-pairs, goat satellite II is present in the genome primarily in the form of 2100 base-pair trimers. Unequal crossing-over between repeat units has produced an oligomer series, whose oligomers gradually decrease in copy number the further they are in molecular weight from the trimer. The trimer population is much more uniform than the monomer population, as most trimers have similar restriction patterns, whereas their component monomers differ considerably in their restriction properties. This heterogeneity was confirmed by cross-hybridization of the different monomers of a cloned trimer. Here, heterologous hybrids were much less stable than the homologous hybrids. Attempts were made to simulate such an oligomer series by computer, using a longitudinal (saltatory), and two horizontal (unequal crossover and drive) models. Simulations of both the saltatory and drive mechanisms could produce the oligomer series in approximately the observed ratios, but only the former could simultaneously produce other restriction properties of this sequence family. This was because the other restriction sites were in a different (monomer) register, and it is difficult for a drive model promoting traits in only one register to fix properties in different registers. The unequal crossover model proposed by Smith (1973, 1976) generally produced homogeneous arrays from heterogeneous arrays, but higher-order repeat structures could be produced when the efficiency of crossing-over between different monomer types was reduced. In most of these arrays, the dimer was the predominant oligomer, but in approximately 10% the trimer was predominant. Since the unequal crossover model produces dimeric arrays with high frequency given appropriate conditions, it is an attractive model for explaining the production of satellite DNAs whose structure has evolved through a series of doublings, such as mouse major satellite DNA and the "alphoid" satellite sequences of primates. Other factors necessary for this model to work are generally considered to be natural components of the speciation process. It is therefore suggested that, although the saltatory model conforms most closely to the observed structure of goat satellite II, this particular satellite DNA may represent one of the few cases when the unequal crossover mechanism does not give rise to a dimeric structure.

Sequence and evolution of related bovine and caprine satellite DNAs. Identification of a short DNA sequence potentially involved in satellite DNA amplification.

The satellite II DNAs of the domestic ox Bos taurus and sheep Ovis aries have been sequenced, and that of the domestic goat Capra hircus partially sequenced. All three are related, and consist of repeat units of about 700 base-pairs. There is no evidence of internal repetition within these repeat units. When matched for maximum homology, the goat and sheep sequences show 83% homology, whereas the ox and sheep sequences share only 70% homology. Factors contributing to the uncertainty of the exact homology between these sequences are discussed, but the results are nevertheless consistent with their progenitor sequence being present in the common ancestor of cattle and sheep. Goat satellite II DNA is shown to contain another, unrelated, tandemly repeated sequence, which is composed of 22 base-pair repeat units. Both this sequence and a region of ox satellite II share good homology with the 11 base-pair progenitor sequence of ox 1.706 g/cm3 satellite DNA. It is suggested that this shared sequence could play a role in bovine satellite DNA amplification.

Isolation and characterization of three cloned fragments of human DNA coding for tRNAs and small nuclear RNA U1.

Employing a human fetal liver library in lambda Charon 4A phage vector, we have isolated and characterized three clones of human DNA containing genes for tRNAs. One clone contains at least three tRNA genes (tRNALys, tRNAGln and tRNALeu) within 2 kb of each other. The other two clones contain two different single genes for tRNAAsn. One of these latter two DNAs also contains a gene for U1 small nuclear RNA.

Comparative structure and evolution of goat and sheep satellite I DNAs.

The satellite I DNAs of domestic goat (Capra hircus) and domestic sheep (Ovis aries) have been studied using molecular hybridisation and restriction enzyme analysis. Both satellite DNAs are composed of repeat units of 820 base pairs in length, but their restriction maps, although similar, differ in certain respects. Thus the majority of sheep satellite I repeat units have two EcoRI sites and one AluI site, whereas the majority of goat satellite I repeat units have one EcoRI site and two AluI sites. The sheep satellite I repeat units with the two EcoRI sites are much more homogeneous than the repeats forming the remainder of the satellite, as judged by the difference in the melting temperatures of native and reassociated molecules. DNAs from species of wild sheep and goats were screened for the presence of these repeat units, and they appear to have been amplified during the radiation of the Ovis genus. Goat satellite I is composed of a single sequence type which has changed through base substitution until the sequence now shows considerable heterogeneity. It is proposed that the major sequence types of these two satellite DNAs were amplified by different saltatory replication events at different times in the evolution of the group.

Specific arrangements of human satellite III DNA sequences in human chromosomes.

DNA was extracted from various rodent-human somatic cell hybrids that contained single or a few human chromosomes. These DNAs were examined by a combination of restriction endonuclease digestion, gel electrophoresis, and filter hybridisation to radioactive satellite DNA probes following transfer of the denatured restriction fragments from a gel to a nitrocellulose filter. In this way the arrangement of sequences homologous to human satellite III were examined on human chromosomes 1, 7, 11, 15, 22 and X. It was found that the distribution of restriction endonuclease sites within satellite III DNA is different on different chromosomes.

Cytogenetic aspects of phylogeny in the Bovidae. I. G-banding.

An extensive G-banding study of karyotypes of 12 species of Bovidae has been undertaken in an attempt to trace homologies and patterns of evolution of karyotype phenotypes throughout the family. G-banding profiles revealed a considerable degree of chromosome-arm homology throughout the group, which also extended into the related superfamilies, the Giraffoidea and Cervoidea. The conservation of banding patterns in chromosome arms strongly indicates that Robertsonian translocation type rearrangements have provided the major source of interspecies karyotype differences, with inversions and reciprocal and tandem translocations providing relatively minor contributions. Examples of individuals carrying newly arisen Robertsonian translocations are not infrequent, and in one instance there was evidence that two similar rearrangements had arisen independently in two species. Despite the extensive changes in karyotype organization, subfamilies within the Bovidae were characterized by the presence of common rearrangements, and those involving autosomal pairs 11 and 12 of the ox, as well as the X chromosome, separate the Bovinae from the Caprinae and Hippotraginae.

Cytogenetic aspects of phylogeny in the Bovidae. II. C-banding.

Constitutive heterochromatin in the Bovidae, as revealed by C-banding, was mostly located in the centromeric regions. Considerable variation was, however, evident in the size of the C-bands both within and between subfamilies. Some evidence was found for a reduction in the amount of centromeric heterochromatin in bi-armed relative to acrocentric autosomes, and these findings are discussed in relation to karyotype evolution in the group.

Relative DNA contents of somatic nuclei of ox, sheep and goat.

Diploid ox nuclei contain about 14% more DNA than nuclei from sheep of the same sex. Goat nuclei have a similar DNA content to those of sheep. In view of the similar chromosome banding patterns in these species, it appears that chromosome evolution must have involved numerous minute interstitial deletions of additions of DNA. Although chromosomes which have similar banding patterns in these three species may be regarded as homologous in this respect, and can be regarded as having a common evolutionary origin, they are not homologous for the quantity of their DNA.