The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

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Initial sequencing and analysis of the human genome.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

http://science.sciencemag.org/content/sci/291/5507/1304.full.pdf

The sequence of the human genome.

Prions cause transmissible and genetic neurodegenerative diseases, including scrapie and bovine spongiform encephalopathy of animals and Creutzfeldt-Jakob and Gerstmann-Straussler-Scheinker diseases of humans. Infectious prion particles are composed largely, if not entirely, of an abnormal isoform of the prion protein, which is encoded by a chromosomal gene. A posttranslational process, as yet unidentified, converts the cellular prion protein into an abnormal isoform. Scrapie incubation times, neuropathology, and prion synthesis in transgenic mice are controlled by the prion protein gene. Point mutations in the prion protein genes of animals and humans are genetically linked to development of neuro-degeneration. Transgenic mice expressing mutant prion proteins spontaneously develop neurologic dysfunction and spongiform neuropathology. Understanding prion diseases may advance investigations of other neurodegenerative disorders and of the processes by which neurons differentiate, function for decades, and then grow senescent.

https://science.sciencemag.org/content/sci/252/5012/1515.full.pdf

Molecular biology of prion diseases

In 1992, we described a second-generation genetic linkage map of the human genome. Using 1,267 new microsatellite markers, we now present a new genetic linkage map containing a total of 2,066 (AC)n short tandem repeats, 60% of which show a heterozygosity of over 0.7. Statistical linkage analysis based on the genotyping of eight large CEPH families placed these markers in the 23 linkage groups. The map includes 1,266 intervals and spans a total distance of 3690 centiMorgans (cM). A total of 1,041 markers could be ordered with odds ratios greater than 1000:1. About 56% of this map is at a distance of 1 cM or less from one of its markers.

The 1993-94 Généthon human genetic linkage map.

Much of natural product chemistry concerns a group of compounds known as secondary metabolites. These low-molecular-weight metabolites often have potent physiological activities. Digitalis, morphine and quinine are plant secondary metabolites, whereas penicillin, cephalosporin, ergotrate and the statins are equally well known fungal secondary metabolites. Although chemically diverse, all secondary metabolites are produced by a few common biosynthetic pathways, often in conjunction with morphological development. Recent advances in molecular biology, bioinformatics and comparative genomics have revealed that the genes encoding specific fungal secondary metabolites are clustered and often located near telomeres. In this review, we address some important questions, including which evolutionary pressures led to gene clustering, why closely related species produce different profiles of secondary metabolites, and whether fungal genomics will accelerate the discovery of new pharmacologically active natural products.

Fungal secondary metabolism — from biochemistry to genomics

Terpenoids are the largest, most diverse class of plant natural products and they play numerous functional roles in primary metabolism and in ecological interactions. The first committed step in the formation of the various terpenoid classes is the transformation of the prenyl diphosphate precursors, geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate, to the parent structures of each type catalyzed by the respective monoterpene (C(10)), sesquiterpene (C(15)), and diterpene synthases (C(20)). Over 30 cDNAs encoding plant terpenoid synthases involved in primary and secondary metabolism have been cloned and characterized. Here we describe the isolation and analysis of six genomic clones encoding terpene synthases of conifers, [(-)-pinene (C(10)), (-)-limonene (C(10)), (E)-alpha-bisabolene (C(15)), delta-selinene (C(15)), and abietadiene synthase (C(20)) from Abies grandis and taxadiene synthase (C(20)) from Taxus brevifolia], all of which are involved in natural products biosynthesis. Genome organization (intron number, size, placement and phase, and exon size) of these gymnosperm terpene synthases was compared to eight previously characterized angiosperm terpene synthase genes and to six putative terpene synthase genomic sequences from Arabidopsis thaliana. Three distinct classes of terpene synthase genes were discerned, from which assumed patterns of sequential intron loss and the loss of an unusual internal sequence element suggest that the ancestral terpenoid synthase gene resembled a contemporary conifer diterpene synthase gene in containing at least 12 introns and 13 exons of conserved size. A model presented for the evolutionary history of plant terpene synthases suggests that this superfamily of genes responsible for natural products biosynthesis derived from terpene synthase genes involved in primary metabolism by duplication and divergence in structural and functional specialization. This novel molecular evolutionary approach focused on genes of secondary metabolism may have broad implications for the origins of natural products and for plant phylogenetics in general.

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Genomic Organization of Plant Terpene Synthases and Molecular Evolutionary Implications

Tree killing bark beetles and their vectored fungal pathogens are the most destructive agents of conifer forests worldwide. Conifers defend against attack by the constitutive and inducible production of oleoresin, a complex mixture of mono-, sesqui-, and diterpenoids that accumulates at the wound site to kill invaders and both flush and seal the injury. Although toxic to the bark beetle and fungal pathogen, oleoresin also plays a central role in the chemical ecology of these boring insects, from host selection to pheromone signaling and tritrophic level interactions. The biochemistry of oleoresin terpenoids is reviewed, and the regulation of production of this unusual plant secretion is described in the context of bark beetle infestation dynamics with respect to the function of the turpentine and rosin components. Recent advances in the molecular genetics of terpenoid biosynthesis provide evidence for the evolutionary origins of oleoresin and permit consideration of genetic engineering strategies to improve conifer defenses as a component of modern forest biotechnology.

Defensive resin biosynthesis in conifers

New mutation in scrapie amyloid precursor gene (at codon 178) in Finnish Creutzfeldt-Jakob kindred.

A total of 116,118 basepairs (bp) derived from three cosmids spanning the ERCC1 locus of human chromosome 19q13.3 have been sequenced with automated fluorescence-based sequencers and analysed by polymerase chain reaction amplification and computer methods. The assembled sequence forms two contigs totalling 105,831 bp, which contain a human fosB proto-oncogene, a gene encoding a protein phosphatase, two genes of unknown function and the previously-characterized ERCC1 DNA repair gene. This light band region has a high average density of 1.4 Alu repeats per kilobase. Human chromosome light bands could therefore contain up to 75,000 genes and 1.5 million Alu repeats.

Automated DNA sequencing and analysis of 106 kilobases from human chromosome 19q13.3

Macrocyclic trichothecenes are toxic sesquiterpenoids that are produced by certain fungi and plants. The unique structural features of macrocyclic trichothecenes result in increased toxicity relative to other trichothecene structural types. Here we report the sequences and relative locations of the MRTRI5, MRTRI6, and MRTRI4 genes in the biosynthetic pathway for macrocyclic trichothecenes in Myrothecium roridum. The deduced sequences of the products of MRTRI5 and MRTRI4 display overall identities of 75 and 63%, respectively, with the corresponding proteins in Fusarium sporotrichioides. Based on sequence comparisons, MRTRI5 encodes the enzyme trichodiene synthase, which has been shown to catalyze the first step in the trichothecene pathways of Fusarium and Trichothecium species. MRTRI6 encodes a transcription factor (392 amino acids) required for pathway gene expression, and the predicted MRTRI4 product (533 amino acids) is a cytochrome P450 monooxygenase responsible for the initial oxygenation step in the pathway. The sizes of the predicted products of MRTRI5 and MRTRI4 show good agreement with their apparent counterparts in the Fusarium pathway; however, the protein specified by MRTRI6 is almost twice the size of its putative homolog in F. sporotrichioides. Only the C-terminal 124 residues of MRTRI6, containing the proposed Cys2His2 zinc finger motifs, show significant similarity (65% identity) to the TRI6 sequence in F. sporotrichioides. MRTRI4 can successfully complement a TRI4-mutant in F. sporotrichioides, although the resulting trichothecene profile differed from that observed in wild-type strains. Complemented mutants accumulated low levels of T-2 toxin, in addition to sambucinol, deoxysambucinol, and the pathway intermediates trichothecene and isotrichodiol. Mapping data indicate that the genes of the macrocyclic trichothecene pathway in M. roridum are clustered, but that their organization and orientation differ markedly from those of the trichothecene gene cluster found in F. sporotrichioides. These results show that the biosynthetic pathways for macrocyclic trichothecenes are closely related to other trichothecene pathways and that the evolution of gene clusters for the biosynthesis of natural products in fungi can involve significant rearrangements.

S Trapp

Papers

Genomic Organization of Plant Terpene Synthases and Molecular Evolutionary Implications

Defensive resin biosynthesis in conifers

New mutation in scrapie amyloid precursor gene (at codon 178) in Finnish Creutzfeldt-Jakob kindred.

Automated DNA sequencing and analysis of 106 kilobases from human chromosome 19q13.3

Characterization of the gene cluster for biosynthesis of macrocyclic trichothecenes in Myrothecium roridum