Chromosomal localization and evolutionary age of satellite DNAs of Mustelidae

DNA reassociation kinetics were studied in the European mink (Mustela lutreola), the American mink (M. vison), the marbled polecat (Vormela peregusna). Variation in DNA quantity and heterochromatin amount occurs in connection with changes in the size of all kinetic fractions. Moderately repetitive genome component is the most variable in these three species. Cryptic CsCl satellite of the stoat (M. erminea), Ag+/Cs2SO4 satellites of the M. vison, V. peregusna were used for in situ homo- and heterologous hybridizations. Satellite DNAs revealed may be classified for the evolution age and chromosomal location type. More ancient satellite DNAs were dispersed in carnivors or mammalian genomes. Mustelids' specific satellites are concentrated in heterochromatic chromosome regions. The evolutionary implications of these findings are discussed.     

Chromosomal localization of seven HSA3q13-->q23 NotI linking clones on chicken microchromosomes: orthology of GGA14 and GGA15 to a gene-rich region of HSA3

Double-color fluorescence in situ hybridization was performed on chicken chromosomes using seven unique clones from the human chromosome 3-specific NotI linking libraries. Six of them (NL1-097, NL2-092, NL2-230, NLM-007, NLM-118, and NLM-196) were located on the same chicken microchromosome and NL1-290 on another. Two chicken microchromosome GGA15-specific BAC clones, JE024F14 containing the IGVPS gene and JE020G17 containing the ALDH1A1 gene, were cytogenetically mapped to the same microchromosome that carried the six NotI linking clones, allowing identification of this chromosome as GGA15. Two GGA14-specific clones, JE027C23 and JE014E08 containing the HBA gene cluster, were co-localized on the same microchromosome as NL1-290, suggesting that this chromosome was GGA14. The results indicated that the human chromosomal region HSA3q13-->q23 is likely to be orthologous to GGA15 and GGA14. The breakpoint of evolutionary conservation of human and chicken chromosomes was detected on HSA3q13.3-->q23 between NL1-290, on the one hand, and six other NotI clones, on the other hand. Considering the available chicken-human comparative mapping data, another breakpoint appears to exist between the above NotI loci and four other genes, TFRC, EIF4A2, SKIL and DHX36 located on HSA3q24-->qter and GGA9. Based on human sequences within the NotI clones, localization of the six new chicken coding sequences orthologous to the human/rodent genes was suggested to be on GGA15 and one on GGA14. Microchromosomal location of seven NotI clones from the HSA3q21 T-band region can be considered as evidence in support of our hypothesis about the functional analogy of mammalian T-bands and avian microchromosomes.     

The heat shock genes in theDrosophila montium subgroup: Chromosomal localization and evolutionary implications

The hsp70, hsp83, hsrω, and the small heat shock protein genes were mapped on the polytene chromosomes of six species, representative of the geographical distribution of the Drosophila montium subgroup of the melanogaster species group. In addition, based on hybridization conditions, the putative locus of the hsp68 gene is given. In contrast to the situation in the melanogaster subgroup species, the hsp70 locus is single in the montium species. The hsp83, hsrωand the small hsp loci are also single in the montium genomes studied here, a common feature of all Drosophila species. Among the hsp genes studied, the small hsp genes and the hsrω-homologous sequences exhibit a higher degree of divergence between the melanogaster and the montium subgroups. Our results support the idea that the montium subgroup species has a genome organization closer to that of the common ancestor compared with the melanogaster subgroup species. Received: 25 July 1995; in revised form: 31 January 1996 / Accepted: 1 April 1996     

Chromosomal distribution, localization and expression of the human endogenous retrovirus ERV9

ERV9 is a class I family of human endogenous retroviral sequences. Somatic cell hybrid genomic hybridization experiments using a mono-chromosomal panel indicate the presence of approximately 120 ERV9 loci in the human genome distributed on most chromosomes. Fluorescence in situ hybridization (FISH) using an ERV9 cDNA probe containing gag, pol and env sequences, verified this observation and a consistent signal was found at the chromosome region 11q13.3-->q13.5. By analysis of a panel of radiation hybrids, an ERV9 locus was mapped to within a 300-kbp region at the chromosome site 11q13. The marker cCLGW567 and the locus MAP3K11/D11S546 centromeric and telomeric flanked it, respectively. Northern blot analysis, using an ERV9 LTR probe, indicated that most normal tissues examined expressed low abundant ERV9 LTR driven mRNAs of various sizes. The most prominent expression was found in adrenal glands and testis. However, the level of expression varied in the same tissues among different individuals indicating that ERV9 mRNA expression probably is inducible in certain tissues or at various cell stages.     

Mammalian milk glycomes: Connecting the dots between evolutionary conservation and biosynthetic pathways

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Investigations on the evolutionary conservation of PCSK9 reveal a functionally important protrusion

Proprotein convertase subtilisin/kexin type 9 (PCSK9) interferes with the recycling of low-density lipoprotein (LDL) receptor (LDLR). This leads to LDLR degradation and reduced cellular uptake of plasma LDL. Naturally occurring human PCSK9 loss-of-function mutations are associated with low levels of plasma LDL cholesterol and a reduced risk of coronary heart disease. PCSK9 gain-of-function mutations result in lower LDL clearance and increased risk of atherosclerosis. The exact mechanism by which PCSK9 disrupts the normal recycling of LDLR remains to be determined. In this study, we have assembled homologs of human PCSK9 from 20 vertebrates, a cephalochordate and mollusks in order to search for conserved regions of PCSK9 that may be important for the PCSK9-mediated degradation of LDLR. We found a large, conserved protrusion on the surface of the PCSK9 catalytic domain and have performed site-directed mutagenesis experiments for 13 residues on this protrusion. A cluster of residues that is important for the degradation of LDLR by PCSK9 was identified. Another cluster of residues, at the opposite end of the conserved protrusion, appears to be involved in the physical interaction with a putative inhibitor of PCSK9. This study identifies the residues, sequence segments and surface patches of PCSK9 that are under strong purifying selection and provides important information for future studies of PCSK9 mutants and for investigations on the function of this regulator of cholesterol homeostasis.     

HSA4 and GGA4: remarkable conservation despite 300-Myr divergence

The chicken (GGA) and human (HSA) genomes diverged around 300-350 Myr ago. Due to this large phylogenetic distance, significant synteny conservation has not been anticipated between the genomes of the two species. However, Zoo-FISH with HSA4 chromosome-specific paint on chicken metaphase chromosomes shows that the human chromosome corresponds largely to the GGA4cen-->q26 region. Comparative gene mapping data in the two species, though limited, provide strong support for these observations. The findings, together with the very recently published data on HSA9-GGAZ and HSA12-GGA1, show that some large chromosomal segments share conserved synteny in the two species. These syntenies are considerably disrupted in the mouse. This makes us believe that despite very early divergence, parts of the human and chicken genomes are more conserved than those of human and mouse, which radiated only 100-120 Myr ago. Moreover, the HSA4-GGA4q correspondence points to a "candidate" chromosome from the karyotype of a mammal-bird ancestor. The findings are thus a small but important step toward understanding the evolution of the two genomes.     

Chromosomal localization of the human c-fms oncogene

A molecular probe was prepared with specificity for the human cellular homologue of transforming sequences represented within the McDonough strain of feline sarcoma virus (v-fms). By analysis of a series of mouse-human somatic cell hybrids containing variable complements of human chromosomes it was possible to assign this human oncogene, designated c-fms, to chromosome 5. Regional localization of c-fms to band q34 on chromosome 5 was accomplished by analysis of Chinese hamster-human cell hybrids containing as their only human components, terminal and interstitial deleted forms of chromosome 5. The localization of c-fms to chromosome 5 (q34) is of interest in view of reports of a specific, apparently interstitial, deletion involving approximately two thirds of the q arm of chromosome 5 in acute myelogenous leukemia cells.     

The DNA sequence of the 5' flanking region of the human beta-globin gene: evolutionary conservation and polymorphic differences.

We have determined the DNA sequence of a 1464 bp segment immediately flanking the 5' side of the human beta-globin gene. The sequence shows little similarity to the corresponding regions of the epsilon- or gamma-globin genes. There is about 75% homology, however, between the 5' extragenic regions of the beta-globin genes of man, goat and rabbit respectively. The mouse beta minor globin gene, but not the mouse beta major globin gene, also shares this extensive homology. A short segment of simple sequence DNA is found from about 1418 to 1388 bp upstream from the human beta-globin gene which consists of repeats of the sequence (TTTTA). Similar DNA sequences are also found at several sites in the large intron of the beta-globin gene. We have compared the DNA sequence of the 5' extragenic region of the normal beta-globin gene with the same segment of the beta-globin gene of a patient with beta thalassaemia. Of the two nucleotide differences observed, one generates a polymorphic HinfI site present 990 bp upstream from the beta-globin gene in the thalassaemic beta-globin and absent in the normal gene. A second beta thalassemic beta-globin gene which has the same molecular defect as the above mentioned case, however, lacks this HinfI site. It is therefore not yet clear whether this HinfI site will have any value in prenatal diagnosis of beta thalassaemia.     

Structure, chromosomal localization and evolutionary conservation of the gene encoding human U1 snRNP-specific A protein.

Three specific proteins, called A, 70K and C, are present in the U1 small nuclear ribonucleoprotein (snRNP) particle, in addition to the common proteins. The human U1 snRNP-specific A protein is, apart from a proline-rich region, highly similar to the U2 snRNP-specific protein B". To examine the homologous regions at the genomic level, we isolated and characterized the human U1-A gene. The human U1-A protein appears to be encoded by a single-copy gene and its locus has been mapped to the q arm of chromosome 19. The gene, about 14-16 kb in length, consists of six exons. The regions homologous to the U2-B" gene are not limited to single exons and are mostly not confined by exon-exon junctions in the corresponding U1-A mRNA. However, the proline-rich region of U1-A, absent in U2-B", is encoded by a single exon, suggesting a specific function for this domain of U1-A. The region of the cap site and upstream sequences contain interesting similarities to the promoter region of other snRNP protein-encoding genes and several housekeeping genes, in particular the vertebrate ribosomal protein-encoding genes. Hybridization experiments with various vertebrate genomic DNAs revealed that U1-A sequences are evolutionarily conserved in all tested vertebrate genomes, except for chicken, duck and pigeon. The divergence of these avian genomes is probably typical for the class of birds.     

The 5'' region of the p53 gene: evolutionary conservation and evidence for a negative regulatory element.

The 5' regions of the mouse, rat and human functional p53 genes were isolated and analysed. All three genes possess a non-coding exon, comprising exclusively 5' untranslated sequences. This exon contains extensive diad symmetry near the 5' end of p53 mRNA, possibly allowing for the formation of a stable hairpin structure in this mRNA. The nucleotide sequence within this hairpin element is highly conserved among the species. A DNA stretch of 225 bp preceding the p53 mRNA cap site possesses distinct promoter activity when assayed in the CAT system. However, this activity is practically abolished when further upstream p53 sequences (approximately 120 bp) are included in front of the CAT gene. This suggests that the control of p53 gene expression is complex and involves a negative regulatory element.