Comparative sequence analysis of the PRKAG3 region between human and pig: evolution of repetitive sequences and potential new exons.

The PRKAG3 gene encodes the gamma3 chain of AMP-activated protein kinase (AMPK). A non-conservative missense mutation in the PRKAG3 gene causes a dominant phenotype involving abnormally high glycogen content in pig skeletal muscle. We have determined >126 kb (in 13 contigs) of porcine genomic sequence surrounding the PRKAG3 gene and the corresponding mouse region covering the gene. A comparison of these PRKAG3 sequences and the human sequence was conducted and used to predict evolutionarily conserved regions, including regulatory regions. A comparison of the human genomic sequence and a porcine BAC sequence containing the PRKAG3 gene, revealed a conserved organization and the presence of three additional genes, CYP27A1 (cytochrome P450, family 27, subfamily A, polypeptide 1), STK36 (Serine Threonine Kinase 36), and the homolog of the unidentified human mRNA KIAA0173. Interspersed repetitive elements constituted 51.4 and 38.6% of this genomic region in human and pig, respectively. We were able to reliably align 12.6 kb of orthologous repeats shared between pig and human and these showed an average sequence identity of 72.4%. Our analysis revealed that the human KIAA0173 gene harbors alternative 5' untranslated exons originating from repetitive elements. This provides an obvious example how transposable elements may affect gene evolution.

A transcriptional roadmap to wood formation.

The large vascular meristem of poplar trees with its highly organized secondary xylem enables the boundaries between different developmental zones to be easily distinguished. This property of wood-forming tissues allowed us to determine a unique tissue-specific transcript profile for a well defined developmental gradient. RNA was prepared from different developmental stages of xylogenesis for DNA microarray analysis by using a hybrid aspen unigene set consisting of 2,995 expressed sequence tags. The analysis revealed that the genes encoding lignin and cellulose biosynthetic enzymes, as well as a number of transcription factors and other potential regulators of xylogenesis, are under strict developmental stage-specific transcriptional regulation.

Sex chromosomal transposable element accumulation and male-driven substitutional evolution in humans.

We sequenced the genomic region containing the human Y-linked zinc finger gene (ZFY). Comparison of ZFY to the related region on the X chromosome (ZFX) and to autosomal sequences reveals a significant accumulation of transposable elements on the sex chromosomes. In addition, five times as many retroviruslike elements (RLEs) are present in the ZFY region as in the ZFX region. Thus, transposable elements accumulate more rapidly on the sex chromosomes, and the insertion of RLEs may occur more frequently in the male than in the female germ line. When the accumulation of substitutions in Alu elements was analyzed, it was found that the Alu elements at the Y-chromosomal locus diverged significantly faster than those at the X-chromosomal locus, whereas the divergence of autosomal Alu elements was intermediate. The male-to-female mutation rate ratio was estimated to be 2.5.

Phylogenetic history of hominoid DRB loci and alleles inferred from intron sequences.

The evolutionary relationships among the MHC class II DRB4, DRB5 and DRB6 loci as well as the allelic lineages and alleles of the DRB1 locus were studied based on intron 1 and intron 2 sequences from humans, chimpanzee (Pan troglodytes), bonobo (Pan paniscus) and gorilla (Gorilla gorilla). The phylogenetic trees for these sequences indicate that most of the DRB1 allelic lineages predate the separation of the hominoid species studied, consistent with previous analysis of the coding sequences of these lineages. However, the intron sequence variation among alleles within DRB1 allelic lineages is very limited, consistent with the notion that the majority of the contemporary alleles have been generated within the last 250,000 years. The clustering of the DRB1 allelic lineages *08 and *12 with *03 supports a common ancestry for the DR8 and DR52 haplotypes. Similarly, the clustering of DRB1 allelic lineages *15 and *01 with the DRB3 locus is consistent with a common ancestry for the DR1 and DR51 haplotypes. Two cases of recombination around the second exon were observed: 1) the HLA-DRB6 locus appears to have been generated through a recombination between a DRB5 allele and an ancestral DRB6 allele, and 2) the gorilla sequence Gogo-DRB1 *03 appears to have been generated through a recombination between the DRB3 locus and an allele from the DRB1 *03 allelic lineage. The nucleotide substitution rate of DRB introns was estimated to 0.85-1.63 x 10(-9) per site per year, based on comparisons between the most closely related sequences from different hominoid species. This estimate is similar to the substitution rate for other intronic regions of the primate genome.

Tracing the origin of HLA-DRB1 alleles by microsatellite polymorphism.

We analyzed the origin of allelic diversity at the class II HLA-DRB1 locus, using a complex microsatellite located in intron 2, close to the polymorphic second exon. A phylogenetic analysis of human, gorilla, and chimpanzee DRB1 sequences indicated that the structure of the microsatellite has evolved, primarily by point mutations, from a putative ancestral (GT)x(GA)y-complex-dinucleotide repeat. In all contemporary DRB1 allelic lineages, with the exception of the human *04 and the gorilla *08 lineages, the (GA)y repeat is interrupted, often by a G-->C substitution. In general, the length of the 3' (GA)y repeat correlates with the allelic lineage and thus evolves more slowly than a middle (GA)z repeat, whose length correlates with specific alleles within the lineage. Comparison of the microsatellite sequence from 30 human DRB1 alleles showed the longer 5' (GT)x to be more variable than the shorter middle (GA)z and 3' (GA)y repeats. Analysis of multiple samples with the same exon sequence, derived from different continents, showed that the 5' (GT)x repeat evolves more rapidly than the middle (GA)z and the 3' (GA)y repeats, which is consistent with findings of a higher mutation rate for longer tracts. The microsatellite-repeat-length variation was used to trace the origin of new DRB1 alleles, such as the new *08 alleles found in the Cayapa people of Ecuador and the Ticuna people of Brazil.

Sexing of human and other primate DNA.

We present a rapid and simple method for the sexing of human and other primate DNA. Homologous introns from the X- and Y-linked zinc finger protein genes (ZFX and ZFY) are PCR amplified with one primer pair. An Alu element insertion in the ZFX intron results in a size difference in the amplicons which is resolved by agarose gel electrophoresis.

Molecular genetics of renal cell carcinoma.

Renal cell carcinoma (RCC) is an important clinical problem for which an effective treatment has yet to be developed. Importantly, the 5-year survival is below 50%. A better understanding of the underlying biological mechanisms could result in improvements in the prevention and treatment of this disease. The molecular mapping of chromosomal losses in renal cell cancer together with increased resolution of the human gene map will provide targets for therapeutic approaches. In this review, I summarize what is known regarding some tumor suppressor genes and candidate tumor suppressor genes in RCC, with reference to their location and expression.

NotI jumping and linking clones as a tool for genome mapping and analysis of chromosome rearrangements in different tumors.

Long-range restriction site maps are of central importance for mapping the human genome. The use of clones from linking and jumping libraries for genome mapping offers a promising alternative to the laborious procedures used up until now. In the present review, this research field is analyzed with particular emphasis on the implementation of a shot-gun sequencing strategy for genome mapping and the use of NotI linking clones for analysis of rearrangements in tumors and tumor cell lines.

New strategy for mapping the human genome based on a novel procedure for construction of jumping libraries.

A novel procedure for construction of jumping libraries is described. The essential features of this procedure are as follows: (1) two diphasmid vectors (lambda SK17 and lambda SK22) are simultaneously used in the library construction to improve representativity, (2) a partial filling-in reaction is used to eliminate cloning of artifactual jumping clones and to obviate the need for a selectable marker. The procedure has been used to construct a representative human NotI jumping library (220,000 independent recombinant clones) from the lymphoblastoid cell line CBMI-Ral-STO, which features a low level of methylation of its resident EBV genomes. A human chromosome 3-specific NotI jumping library (500,000 independent recombinant clones) from the human chromosome 3 x mouse hybrid cell line MCH 903.1 has also been constructed. Of these recombinant clones 50-80% represent jumps to the neighboring cleavable NotI site. With our previously published method for construction of linking libraries this procedure makes a new genome mapping strategy feasible. This strategy includes the determination of tagging sequences adjacent to NotI sites in random linking and jumping clones. Special features of the lambda SK17 and lambda SK22 vectors facilitate such sequencing. The STS (sequence tagged site) information obtained can be assembled by computer into a map representing the linear order of the NotI sites for a chromosome or for the entire genome. The computerized mapping data can be used to retrieve clones near a region of interest. The corresponding clones can be obtained from the panel of original clones, or necessary probes can be made from genomic DNA by PCR.

Involvement of 3p deletions in sporadic and hereditary forms of renal cell carcinoma.

Deletions of the short arm of chromosome 3 and associated allele losses have been reported in the majority of sporadic renal cell carcinomas (RCC). On the basis of the combined cytogenetic and molecular data, it is reasonable to assume that a putative RCC locus, which contributes to tumor development by its loss, is located telomerically of the D3F15S2 site. Using H3E4, a D3F15S2-specific probe, we have isolated a cDNA clone (cl.4-2), and a sequence comparison revealed that the cDNA clone corresponds to the human acyl-peptide hydrolase gene. The gene is fairly universally expressed, but in RCC biopsies its expression is severely reduced, compared to the normal kidney. Cl.4-2 was used for in situ hybridization on metaphase chromosomes prepared from an Epstein-Barr virus (EBV) transformed lymphoblastoid cell line, derived from a t(3;8) (p14.2;q24.1) carrying member of the RCC family described by Cohen et al. in 1979 (N Engl J Med: 301:592-595). Carriers of this translocation regularly develop RCC by middle age. We now report that D3F15S2 is localized on the telomeric side of the constitutional breakpoint, in 3p21. The region of 3p affected by this familial translocation is thus not identical with the region of 3p most frequently deleted in sporadic RCC.

The gene from the short arm of chromosome 3, at D3F15S2, frequently deleted in renal cell carcinoma, encodes acylpeptide hydrolase.

Loss or inactivation of a gene on the short arm of chromosome 3 may contribute to the genesis of renal cell carcinoma. A gene that corresponds to the most frequently lost RFLP site (D3F15S2) is expressed in a variety of human tissues, and at a particularly high level in the kidney. Its expression is markedly reduced in renal cell carcinoma. A database search showed that the gene product is closely related to or identical with acylpeptide hydrolase. The nucleotide identity between the rat acylpeptide hydrolase and the human gene at D3F15S2 is 88%, compatible with normal species differences. It is therefore likely that the human gene product is acylpeptide hydrolase. The renal cell carcinoma is then associated with a decrease of acylpeptide hydrolase activity. The gene may represent a tumor suppressor gene, whose loss contributes to the development of renal cell carcinoma. It might be speculated that it could act e.g. by affecting the activity of a small acetylated growth factor. Alternatively, its decreased expression may merely reflect the impairment of differentiation in RCC, compared to normal kidney. Loss of a linked but irrelevant gene by the 3p deletion is another possibility.

Structure of adsorbed fibrinogen obtained by scanning force microscopy.

It is shown that scanning force microscopy (SFM), operated in the attractive mode, can be used to obtain high resolution pictures of adsorbed fibrinogen molecules on solid surfaces, without the need for staining or special microscope grids. SFM also reveals the three-dimensional structure of the adsorbed molecules. Two forms of adsorbed fibrinogen are demonstrated on hydrophobic silicone dioxide surfaces: a trinodular about 60 nm long and a globular with about a 40 nm diameter. Polymeric networks formed after storage of the surface with adsorbed fibrinogen in PBS for 11 days are also shown. The SFM-results for the trinodular structure suggest the existence of loops or peptide chains extending outside the basic structure of the fibrinogen molecule.

The most frequently lost allelic site in human renal cell carcinoma (D3F15S2) on the short arm of chromosome 3 has homologous sequences on rat chromosome 8.

It has previously been shown that human chromosome 3 has banding homology to rat chromosome 8. We have previously isolated a cDNA from the D3F15S2 region and designated the gene as RIK. In the present study, we localized the homolog of this gene to rat chromosome 8.

Construction of a human chromosome 3 specific NotI linking library using a novel cloning procedure.

Two new diphasmid vectors (lambda SK17 and SK22) and a novel procedure to construct linking libraries are described. A partial filling-in reaction provides counter-selection against false linking clones in the library, and obviates the need for supF selection. The diphasmid vectors, in combination with the novel selection procedure, have been used to construct a chromosome 3 specific NotI linking library from a human chromosome 3/mouse microcell hybrid cell line. The application of the new vectors and the strong biochemical and biological selections resulted in a library of 60,000 NotI linking clones. As practically all of them are real NotI linking clones (no false recombinants) the library represents approximately 3,000 human recombinants (equal to 10-15 genomic equivalents of chromosome 3). Previously published methods for construction of linking libraries are compared with the procedure described in the present paper. The advantages of the new vectors and the novel protocol are discussed.

A gene near the D3F15S2 site on 3p is expressed in normal human kidney but not or only at a severely reduced level in 11 of 15 primary renal cell carcinomas (RCC).

Renal Cell Carcinoma (RCC) has been associated with the loss of heterozygosity at several loci on the short arm of chromosome 3 (3p). We have previously found that one of these loci, D3F15S2 (pH3H2) was lost in 76% of the tumor cells derived from heterozygous donors (Kovacs, G., Erlandsson, R., Boldog, F., Ingvarrsson, S., Müller-Brechlin, R., Klein, G. & Sümegi, J. (1988), Proc. Natl. Acad. Sci., 85, 1571-5). More recently we have identified a putative CpG island in the vicinity of D3F15S2, suggesting that DNA sequences in or around this site may have coding potential (Boldog, F., Erlandsson, R., Klein, G. & Sümegi, J. (1989). Cancer Genet. Cytogenet., 42, 295-306). The screening of a human placenta cDNA library with DNA probes derived from D3F15S2 has led to the isolation of several cDNA clones. They identified a 2.9 Kb long message in human placenta and kidney. In total RNA from 11 of 15 primary RCCs the gene expression was reduced to less than 20% compared to eight normal kidneys. This low level of expression may be due to contaminating normal tissue. In the remaining 4 tumors the expression varied from 24-51% compared to normal kidney. To facilitate reference, the gene was provisionally designated as 'RIK'. It was expressed in the HEK 293, one osteosarcoma (HOS), two carcinoma (COLO320 and QDMT), and three Burkitt lymphoma lines (BL2, BL29 and BL31). It was not expressed in one Burkitt lymphoma (DG75) and two EBV transformed lymphoblastoid cell lines (LCL) (NAD-20 and Cherry).

Long-range restriction enzyme maps of DNF15S2, D3S2, and c-raf1 loci on the short arm of human chromosome 3.

Physical maps have been constructed around loci DNF15S2, D3S2, and c-raf1 on the short arm of human chromosome 3 using pulsed field gradient gel electrophoresis. The normal restriction pattern has not been altered by a t(3;8)(p14.2;q24.1) characteristic for a hereditary form of renal cell carcinoma, indicating that the breakpoint itself is not included in any of the mapped areas. We have found a CpG island within the DNF15S2 locus, suggesting the presence of a functional gene in the region.

Do human renal cell carcinomas arise by a double-loss mechanism?

Combined consideration of a constitutional t(3;8) that was regularly associated with renal cell carcinoma (RCC) within a large family and cytogenetic and restriction fragment length polymorphism studies on sporadic RCC has led to the tentative conclusion that RCC may arise by a similar double-loss mechanism as retinoblastoma and Wilms' tumor. This hypothesis predicts single-hit kinetics for the age distribution of hereditary RCC and two-hit kinetics for sporadic tumors, in analogy with Knudson's original prediction for retinoblastoma and Wilms' tumor. We have compared the age distribution of 51 hereditary and 56 sporadic cases of RCC sampled from the literature. The age-incidence curve of the hereditary RCC is compatible with a single event, whereas the sporadic tumors arise as predicted from a two-hit curve. We therefore suggest that RCC arises by the loss of a recessive cancer gene, probably localized to the short arm of chromosome 3 (in band 3p14.2).

Consistent chromosome 3p deletion and loss of heterozygosity in renal cell carcinoma.

Renal cell carcinoma (RCC) and normal kidney tissues have been examined from 34 patients with sporadic, nonhereditary RCC. Eighteen of the 21 cytogenetically examined tumors (86%) had a detectable anomaly of chromosome arm 3p distal to band 3p11.2-p13, manifested as a deletion, combined with the nonreciprocal translocation of a segment from another chromosome or monosomy 3. Restriction-fragment-length polymorphism analysis showed loss of D1S1 heterozygosity in 16 of the 21 cases (76%). D3S2 heterozygosity was lost in 2 of 11 cases (18%). The variability of the breakpoint between 3p11.2 and 3p13 and the absence of a consistently translocated segment from another chromosome suggests a genetic-loss mechanism, while the activation of a dominant oncogene appears less likely. Together with the previously demonstrated involvement of the 3p14.2 region in a familial case, these findings suggest that RCCs may arise by the deletion of a "recessive cancer gene," as do retinoblastoma and Wilms tumor. The relevant locus must be located on the telomeric side of the D1S1 locus on the short arm of chromosome 3.