Isolation and genomic analysis of the human surf-6 gene: a member of the Surfeit locus

The human Surfeit locus contains at least six tightly clustered genes (Surf-1 to Surf-6) of which five (Surf-1 to Surf-5) have been characterised and found not to share any sequence homology. The organisation and juxtaposition of the Surfeit genes are conserved between human and mouse. The Surf-6 gene that encodes a novel nucleolar-matrix protein with nucleic-acid binding properties has been characterised in mouse. In this work, we have isolated and analysed the human Surf-6 homologue and determined its genomic organisation in the Surfeit locus. The human Surf-6 gene has five exons spread over a distance of 4.3kb and has features of a housekeeping gene being ubiquitously expressed, having its 5' end located within a CpG rich island and lacking a canonical TATA box. The intragenic region between the 3' end of the Surf-5 gene and the 5' end of the Surf-6 gene is 3.2kb and contains a pseudogene of the ribosomal protein gene rpL21. The putative human Surf-6 protein is 361 amino acids long and includes motifs found in both the mouse and fish Surf-6 homologues, which may underlie the functions of Surf-6. Three amino acid polymorphisms have been detected at codons 163, 175 and 311 by SSCP analysis.     

Gene-associated CpG islands in plants as revealed by analyses of genomic sequences

We screened plant genome sequences, primarily from rice and Arabidopsis thaliana, for CpG islands, and identified DNA segments rich in CpG dinucleotides within these sequences. These CpG-rich clusters appeared in the analysed sequences as discrete peaks and occurred at the frequencies of one per 4.7 kb in rice and one per 4.0 kb in A. thaliana. In rice and A. thaliana, most of the CpG-rich clusters were associated with genes, which suggests that these clusters are useful landmarks in genome sequences for identifying genes in plants with small genomes. In contrast, in plants with larger genomes, only a few of the clusters were associated with genes. These plant CpG-rich clusters satisfied the criteria used for identifying human CpG islands, which suggests that these CpG clusters may be regarded as plant CpG islands. The position of each island relative to the 5'-end of its associated gene varied considerably. Genes in the analysed sequences were grouped into five classes according to the position of the CpG islands within their associated genes. A large proportion of the genes belonged to one of two classes, in which a CpG island occurred near the 5'-end of the gene or covered the whole gene region. The position of a plant CpG island within its associated gene appeared to be related to the extent of tissue-specific expression of the gene; the CpG islands of most of the widely expressed rice genes occurred near the 5'-end of the genes.     

Characterization of the human laminin beta2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS).

The laminin beta2 chain is an important constituent of certain kidney and muscle basement membranes. We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin beta2 chain gene (LAMB2) on chromosome 3p21.3-->p21.2, a region paralogous with the chromosome 7q22-->q31 region that contains the laminin beta1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3' end of LAMB2 lies 16 kb from the 5' end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3' end of LAMB2L was similar to exons 8-12 of the laminin beta2 chain gene. The LAMB2L-LAMB2-QARS cluster lies telomeric to the gene encoding the laminin-binding protein dystroglycan (DAG1).     

CpG islands of the X chromosome are gene associated.

Unmethylated CpG rich islands are a feature of vertebrate DNA: they are associated with housekeeping and many tissue specific genes. CpG islands on the active X chromosome of mammals are also unmethylated. However, islands on the inactive X chromosome are heavily methylated. We have identified a CpG island in the 5' region of the G6PD gene, and two islands forty Kb 3' from the G6PD gene, on the human X chromosome. Expression of the G6PD gene is associated with concordant demethylation of all three CpG islands. We have shown that one of the two islands is in the promoter region of a housekeeping gene, GdX. In this paper we show that the second CpG island is also associated with a gene, P3. The P3 gene has no homology to previously described genes. It is a single copy, 4 kb gene, conserved in evolution, and it has the features of a housekeeping two genes is within the CpG island and that sequences in the islands have promoter function.     

Surfeit locus gene homologs are widely distributed in invertebrate genomes.

The mouse Surfeit locus contains six sequence-unrelated genes (Surf-1 to -6) arranged in the tightest gene cluster so far described for mammals. The organization and juxtaposition of five of the Surfeit genes (Surf-1 to -5) are conserved between mammals and birds, and this may reflect a functional or regulatory requirement for the gene clustering. We have undertaken an evolutionary study to determine whether the Surfeit genes are conserved and clustered in invertebrate genomes. Drosophila melanogaster and Caenorhabditis elegans homologs of the mouse Surf-4 gene, which encodes an integral membrane protein associated with the endoplasmic reticulum, have been isolated. The amino acid sequences of the Drosophila and C. elegans homologs are highly conserved in comparison with the mouse Surf-4 protein. In particular, a dilysine motif implicated in endoplasmic reticulum localization of the mouse protein is conserved in the invertebrate homologs. We show that the Drosophila Surf-4 gene, which is transcribed from a TATA-less promoter, is not closely associated with other Drosophila Surfeit gene homologs but rather is located upstream from sequences encoding a homolog of a yeast seryl-tRNA synthetase protein. There are at least two closely linked Surf-3/rpL7a genes or highly polymorphic alleles of a single Surf-3/rpL7a gene in the C. elegans genome. The chromosomal locations of the C. elegans Surf-1, Surf-3/rpL7a, and Surf-4 genes have been determined. In D. melanogaster the Surf-3/rpL7a, Surf-4, and Surf-5 gene homologs and in C. elegans the Surf-1, Surf-3/rpL7a, Surf-4, and Surf-5 gene homologs are located on completely different chromosomes, suggesting that any requirement for the tight clustering of the genes in the Surfeit locus is restricted to vertebrate lineages.     

Complete nucleotide sequence of a functional HLA-DP beta gene and the region between the DP beta 1 and DP alpha 1 genes: comparison of the 5'' ends of HLA class II genes.

The complete nucleotide sequence of an HLA-DP beta 1 gene and part of the adjacent DP alpha 1 gene, up to and including the signal sequence exon, were determined. The sequence of the DP beta 1 gene identified it as the DPw4 allele. The six exons of the DP beta 1 gene spanned over 11,000 bp of sequence. The arrangement of the gene was broadly analogous to genes of other class II beta chains. The beta 1 exon was flanked by introns of over 4 kb. Comparisons with published sequences of cDNA clones indicated that an alternative splice junction, at the 3' end of the gene, is used in at least one allele. Variation in choice of splice junction indicates an additional mechanism for allelic variation in class II genes. The sequence also indicated that the DP beta 1 and DP alpha 1 genes are separated by only 2 kb at their 5' ends. Comparison of the 5' ends of the DP alpha 1 and beta 1 genes with other class II sequences, including the DZ alpha gene, showed conservation of several blocks of sequences thought to be involved in control of expression. Some areas of the introns were partially conserved in the DQ beta gene, and several other intron sequences were homologous to sequences found in other unrelated genes.     

Unusual features of CpG-rich (HTF) islands in the human alpha globin complex: association with non-functional pseudogenes and presence within the 3'' portion of the zeta gene.

We have characterised a cluster of CpG rich (HTF) islands in the alpha-globin complex and report here two unusual features: The human embryonic zeta 2-globin gene is associated with an HTF island within its 3' portion rather than at the 5' end. Furthermore at least two non-functional pseudogenes within the cluster (psi zeta 1 and psi alpha 2) are associated with CpG rich islands.     

Characterization ofAlu family repetitive sequences which flank human β-type globin genes

Heteroduplex mapping has determined the size, location, and orientation of three Alu family sequences from the human beta-type globin gene cluster. Two of these sequences have the same orientation. One (231 bp long) is 2 kb to the 5' side of the B gamma-globin gene and the other (222 bp) is l kb 5' to the pseudo-beta-l-globin gene. The third (300 bp), 3-4 kb 3' to the pseudo-beta-l-globin gene, has the opposite orientation. Their orientations relative to five previously characterized Alu sequences from this cluster have been established. One of these, 2.5 kb 5' to the epsilon-globin gene, was shown by Southern blot hybridization to be similar but not identical to other family members, whereas the region separating it from a neighbouring inverted repeat is not widely distributed in the human genome.     

Molecular cloning and characterization of the human beta-like globin gene cluster

The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.     

Repetitive DNA sequences near three human beta-type globin genes.

Five repetitive DNA sequences, of average length 259 bp, have been identified in the intergenic regions which flank three human beta-tupe globin genes. A pair of inverted repeat sequences, separated by 919 bp, was found 1.0 kb to the 5' side of the epsiln-globin gene. Each contains a homologous Alu I site. Another repetitive sequence, with the same orientation as the inverted repeat sequence closest to the epsilon-globin gene, lies about 2.2 kb to the 5' side of the delta-globin gene. A pair of inverted repeat sequences, with the same relative orientations as the other pair and separated by about 800 bp, was found about 1.5 kb to the 3' side of the beta-globin gene.