A physical map of the apolipoprotein gene cluster on human chromosome 19

Summary We have generated a restriction map around the cloned genes for human apolipoproteins CI, CII, and E by pulsed-field gel analysis. We show that the genes are clustered within an area of about 50 kb on chromosome 19. The genes are all oriented in the same direction, head to tail.     

The structure and evolution of the human salivary proline-rich protein gene family

We present the nucleotide sequences of four members of the six-member human salivary prolinerich protein (PRP) gene family. The four genes are PRB1 and PRB2, which encode basic PRPs, and PRB3 and PRB4, which encode glycosylated PRPs. Each PRB gene is approximately 4.0 kb in length and contains four exons, the third of which is entirely composed of 63-bp tandem repeats and encodes the proline-rich portion of the protein products. Exon 3 contains different numbers of tandem repeats in the different PRB genes. Variation in the numbers of these repeats is also responsible for length variations in different alleles of the PRB genes. We have determined a probable evolutionary history of the human PRP gene family by comparing the nucleotide sequences of the six PRP genes. The present-day six PRP loci probably evolved from a single ancestral gene by four sequential gene duplications, leading to six genes that fall into three subsets, each consisting of two genes. During this evolutionary process, multiple rearrangements and gene conversion occurred mainly in the region from the 3 end of IVS2 and the 3 end of exon 3.     

Nucleotide sequence of gene PBII encoding salivary proline-rich protein P-B

The nucleotide sequence of gene PBII encoding salivary proline-rich protein P-B was determined. PBII is 7.1 kb long and contains 3 exons. PBII exhibits considerable nucleotide sequence homology not only in exons but also in introns with PBI (accession number D89501), the gene whose nucleotide sequence was determined previously [Isemura and Saitoh (1997) J. Biochem. 121, 1025-1030]. PBI and II constitute a gene family distinct from that to which the majority of salivary proline-rich protein ones belong. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number AB031740.     

A physical map of the human APP gene in YACs

Several point mutations within exons 16 and 17 of the amyloid precursor protein (APP) gene have been reported that are associated with Alzheimer's disease in a small number of familial cases. To determine the size of the APP gene and the organization of the exons within human genomic DNA, we have characterized 11 Yeast Artificial Chromosome (YAC), recombinants containing human APP gene sequences. The smallest YAC insert was 125 kb, and the largest was 1.4 Mb. The YACs were screened by polymerase chain reaction amplification of APP exons to determine which of the 18 exons coding for APP770 were present. Four of the YACs (D110G1, D110G6, D110E9, and B142F9) contain all 18 exons and at least part of the promoter. Construction of an overlapping map of the gene with all of the YACs demonstrated that 3 of the 11 YACs were chimeric. The orientation and position of the coding sequence on the map was determined by probing digests of the YAC DNA with exon PCR products and the vector arms. The coding region of the APP gene spans approximately 400 kb of genomic DNA.     

Synthesis and circular dichroism study of the human salivary proline-rich protein IB7.

The solid phase synthesis of a 59 amino acid human salivary protein IB7 has been accomplished using Fmoc strategy. Because the protein contains 25 proline, 13 glycine and 9 glutamine residues the coupling time, piperidine delivery and acetic anhydride reaction time were increased. Yield after HPLC purification was 35%. Circular dichroism studies revealed that about one third of IB7 residues adopted a type II helix secondary structure, as found in collagen helices. The rest of the sequence adopts a random coil secondary structure.     

Secondary structure prediction of human salivary proline-rich proteins

Conformations associated with secondary structure in human salivary proline-rich proteins A (PRPA), C (PRPC), P-D and P-E were predicted by analysis of their respective hydrophobicity profiles by computer programming. Structurally, PRPA and PRPC would present a globular head and a tail that consists of type 3(10) polyproline helices. P-D and P-E would be fibrilar molecules with helical zones of the polyproline 3(10) type. Alternatively for PRPA and PRPC, the head and tail would form one globular domain with the tail folding upon itself at places where random coils occur.     

Isolation,physical map and gene map of mitochondrial DNA from the cryptomonad Pyrenomonas salina

Mitochondrial DNA (mtDNA) from the cryptomonad Pyrenomonas salina was isolated by CsCl-buoyant density centrifugation of whole-cell DNA in the presence of Hoechst dye 33258. mtDNA consists of circular molecules about 47 kb in size as estimated from restriction enzyme analysis. A physical map for six restriction enzymes (Bam HI, Bge I, Eco RI, Pst I, Sac I and Sac I) has been constructed. Genes coding for the small subunit of rRNA, cytochrome oxidase subunits I and II, and apocytochrome b were localized on this map using Southern blot hybridization with heterologous gene probes from Oenothera. Genes for 5S rRNA and NADH dehydrogenase subunit 5 are absent from P. salina mtDNA. The mitochondrial genome, being the first analysed to this extent in chromophytic algae, should be valuable for taxonomic and phylogenetic studies.     

Construction of a physical map of the 45 kb photosynthetic gene cluster of Rhodobacter sphaeroides

1) A number of overlapping clones have been isolated from a Rhodobacter sphaeroides gene bank. Following conjugative gene transfer from Escherichia coli these clones restore a wild type phenotype to several mutants unable to synthesise bacteriochlorophyll. 2) The insert DNA was analysed by restriction mapping and together the clones form the basis of the first restriction map of the 45 kb photosynthetic gene cluster of Rb. sphaeroides. 3) This cluster is bounded on one side by puh A encoding the reaction centre H polypeptide and on the other by the puf operon encoding reaction centre L and M apoproteins and light harvesting LH1 and polypeptides. 4) DNA fragments from the 45 kb cluster were used to probe genomic DNA from other photosynthetic bacteria. Using heterologous hybridisation conditions, a significant degree of homology is shown between Rb. sphaeroides and these other bacteria, suggesting close evolutionary links with Rb. capsulatus in particular.     

A physical map of the human Y-chromosome short arm

A physical map of the Y-chromosome short arm was constructed using DNA probes p19B, Y-286/la5, pZFY, Y-280, and Y-227. These probes hybridize with four NotI fragments of 400 kb (p19B and Y-286/la5), 350 kb, 1.9 Mb, and 3.0 Mb, respectively. The restriction fragments were shown to be adjacent to each other by analysis of NotI partial digests, overlapping restriction fragments, and/or the detection of rearranged restriction fragments in a 46,XX male. The present map covers approximately 5.6 Mb of contiguous DNA of Yp. Previously, the size of the pseudoautosomal region was estimated to be 2.3 Mb, and a 5.3-Mb NotI fragment containing Y-specific repeated DNA was assigned to proximal Yp. These and the present data account for approximately 13 Mb and thus for most of the DNA content of the Y short arm.     

A physical map of the human pseudoautosomal region.

A physical map of the human pseudoautosomal region has been constructed using pulsed field gel electrophoresis and the infrequently cutting restriction enzymes BssHIII, EagI, SstII, NotI, MluI and NruI. This map extends 2.3 Mbp from the telomere to sex-chromosome-specific DNA, includes at least seven CpG islands and locates four genetically mapped loci. Five of the CpG islands are organized into two clusters. One cluster is adjacent to the telomere, the other extends into sex-chromosome-specific DNA. There is congruence between the genetic and physical maps which implies that the frequency of recombination is approximately uniform throughout the DNA.     

A physical map of the human PI and AACT genes

We have used probes from the human genes PI, PIL, and AACT (alpha 1-antitrypsin, alpha 1-antitrypsin-related sequence, and alpha 1-antichymotrypsin) to make a pulsed-field map of the surrounding region of 14q31-32. We have discovered that the PI-PIL gene cluster is only 220 kb away from the AACT gene and that it is orientated in the opposite direction. The comparatively short distance between the genes comes as a surprise given previous estimates of the level of genetic recombination between them.     

A 10-megabase physical map of human Xp21, including the Duchenne muscular dystrophy gene

Using pulsed-field gel electrophoresis and 12 Xp21-derived DNA probes, we have constructed a continuous restriction map spanning more than 4 million base pairs (4 Mbp), including the Duchenne muscular dystrophy gene of more than 2 Mbp. This detailed map is part of a less detailed map spanning 10 Mbp, also spanning the genes for glycerol kinase and congenital adrenal hypoplasia, constructed under electrophoresis conditions which separated DNA fragments in the range 200 to 4000 kbp. DNA from three different tissues was analyzed, and differential methylation was observed.     

Localization of the sapA gene on a physical map of Campylobacter fetus chromosomal DNA

     We constructed a physical map of Campylobacter fetus TK(+) chromosomal DNA digested by either SmaI, SalI, or NotI using pulsed-field gel electrophoresis and Southern hybridization data. The genome size of C. fetus TK(+) is 2016 kb, larger than that reported by the others. To locate the sapA gene, which encodes the surface array protein (SAP), on the physical map, we performed Southern hybridizations with probes based on the conserved region of the sapA gene. The results showed that more than seven copies of the conserved region were present on C. fetus chromosomal DNA and that the sapA gene was located on a limited number of fragments forming a cluster of genes. By comparing fingerprint patterns of strain TK(+) and strain TK(–), which lost the ability to produce SAP during culture on agar medium, an approximately 10 kb deletion was observed in the fragments of strain TK(–). The results of Southern hybridization with two probes, one from the upstream region and the other from the variable region of sapA, suggest that the loss of SAP expression might not be the result of the loss of the sapA gene itself, but only a loss of its control systems. Received: 25 May 1994 / Accepted: 1 September 1994