DNA-PKcs: a T-cell tumour suppressor encoded at the mouse scid locus.

Severe combined immunodeficiency (SCID) mice are defective in their ability to rearrange their variable (V), diversity (D) and joining (J) genetic elements to generate functional immunoglobulin (Ig) and T-cell receptor (TCR) molecules; as a result, they lack mature B and T cells. These mice are highly sensitive to ionizing radiation, suggesting that the product of the scid gene plays a critical role in both V(D)J recombination and DNA double-strand break repair. Recent studies suggest that the SCID defect lies in the gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PK; refs 6-8), a nuclear protein made up of the Ku 70 and Ku 86 subunits as well as the large catalytic subunit, DNA-PKcs. Other reports have implied that the SCID phenotype correlates with nonsense mutations at the extreme 3' end of Prkdc, the DNA-PKcs gene. The identity of the gene remains in doubt, however, because the consequences of genetic inactivation of Prkdc have not been determined. This study shows that complete inactivation of Prkdc in a novel insertional mouse mutant recapitulates the SCID phenotype and that Prkdc and scid are alleic. Significantly, DNA-PKcs null mice demonstrate complete penetrance of thymic lymphoblastic lymphomas, strongly suggesting that Prkdc functions in mice as a T-cell tumour suppressor and, by virtue of its association with DNA repair and recombination, belongs to the 'caretaker' class of tumour-suppressor genes that includes ATM, BRCA1 and BRCA2 (ref. 15).

Frequency and distribution of DNA uptake signal sequences in the Haemophilus influenzae Rd genome.

The naturally transformable, Gram-negative bacterium Haemophilus influenzae Rd preferentially takes up DNA of its own species by recognizing a 9-base pair sequence, 5'-AAGTGCGGT, carried in multiple copies in its chromosome. With the availability of the complete genome sequence, 1465 copies of the 9-base pair uptake site have been identified. Alignment of these sites unexpectedly reveals an extended consensus region of 29 base pairs containing the core 9-base pair region and two downstream 6-base pair A/T-rich regions, each spaced about one helix turn apart. Seventeen percent of the sites are in inverted repeat pairs, many of which are located downstream to gene termini and are capable of forming stem-loop structures in messenger RNA that might function as signals for transcription termination.

Mutation of a mutL homolog in hereditary colon cancer.

Some cases of hereditary nonpolyposis colorectal cancer (HNPCC) are due to alterations in a mutS-related mismatch repair gene. A search of a large database of expressed sequence tags derived from random complementary DNA clones revealed three additional human mismatch repair genes, all related to the bacterial mutL gene. One of these genes (hMLH1) resides on chromosome 3p21, within 1 centimorgan of markers previously linked to cancer susceptibility in HNPCC kindreds. Mutations of hMLH1 that would disrupt the gene product were identified in such kindreds, demonstrating that this gene is responsible for the disease. These results suggest that defects in any of several mismatch repair genes can cause HNPCC.

Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.

An approach for genome analysis based on sequencing and assembly of unselected pieces of DNA from the whole chromosome has been applied to obtain the complete nucleotide sequence (1,830,137 base pairs) of the genome from the bacterium Haemophilus influenzae Rd. This approach eliminates the need for initial mapping efforts and is therefore applicable to the vast array of microbial species for which genome maps are unavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase accession number L42023) represents the only complete genome sequence from a free-living organism.

Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii.

The complete 1.66-megabase pair genome sequence of an autotrophic archaeon, Methanococcus jannaschii, and its 58- and 16-kilobase pair extrachromosomal elements have been determined by whole-genome random sequencing. A total of 1738 predicted protein-coding genes were identified; however, only a minority of these (38 percent) could be assigned a putative cellular role with high confidence. Although the majority of genes related to energy production, cell division, and metabolism in M. jannaschii are most similar to those found in Bacteria, most of the genes involved in transcription, translation, and replication in M. jannaschii are more similar to those found in Eukaryotes.

The minimal gene complement of Mycoplasma genitalium.

The complete nucleotide sequence (580,070 base pairs) of the Mycoplasma genitalium genome, the smallest known genome of any free-living organism, has been determined by whole-genome random sequencing and assembly. A total of only 470 predicted coding regions were identified that include genes required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. Comparison of this genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.

Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1.

The complete genome sequence of the radiation-resistant bacterium Deinococcus radiodurans R1 is composed of two chromosomes (2,648,638 and 412,348 base pairs), a megaplasmid (177,466 base pairs), and a small plasmid (45,704 base pairs), yielding a total genome of 3,284, 156 base pairs. Multiple components distributed on the chromosomes and megaplasmid that contribute to the ability of D. radiodurans to survive under conditions of starvation, oxidative stress, and high amounts of DNA damage were identified. Deinococcus radiodurans represents an organism in which all systems for DNA repair, DNA damage export, desiccation and starvation recovery, and genetic redundancy are present in one cell.

Genome data: what do we learn?

Genome sequence information has continued to accumulate at a spectacular pace during the past year. Details of the sequence and gene content of human chromosome 22 were published. The sequencing and annotation of the first two Arabidopsis thaliana chromosomes was completed. The sequence of chromosome 3 from Plasmodium falciparum, the second sequenced malaria chromosome, was reported, as was that of chromosome 1 from Leishmania major. The complete genomic sequences of five microbes were reported. Approaches to using data from completely sequenced microbial genomes in phylogenetic studies are being explored, as is the application of microarrays to whole genome expression analysis.

Evidence that a 90-kDa phosphoprotein, an associated kinase, and a specific phosphatase are involved in the regulation of Cloudman melanoma cell proliferation by insulin.

Proliferation of wild-type Cloudman S91 melanoma cells is inhibited when insulin is included in the culture medium. Using growth inhibition as a selective marker, we isolated variant cell lines that are either resistant to insulin or dependent upon insulin for growth. We have studied the effects of insulin on proliferation by using combined genetic and biochemical approaches. Through a series of genetic hybridization analyses, we have identified three complementation groups and determined that, in general, insulin-sensitivity is dominant to insulin-resistance. Through analyses of in vitro protein phosphorylation reactions, we have identified a protein of approximately 90 kDa (pp90) whose phosphorylation is a function of at least one of the complementation groups. Although pp90 is not phosphorylated in extracts of insulin-resistant variants, it is phosphorylated in extracts of insulin-sensitive hybrids formed between complementing resistant variants. Insulin itself exhibits little or no regulation over the phosphorylation of pp90; rather, the ability to phosphorylate pp90 correlates with the ability of cells to respond to insulin. Migration in NaDodSO4/polyacrylamide gels, solubility characteristics, and divalent cation requirements indicate that pp90 is distinct from the 95-kDa beta-subunit of the insulin receptor. Both pp90 and its associated phosphoprotein kinase are found in 30,000 X g pellets of sonicated cell lysates, whereas a specific pp90 phosphoprotein phosphatase activity is found in 30,000 X g supernatant fractions. Phosphorylation of pp90 occurs at tyrosine and serine residues. Our evidence indicates that the state of phosphorylation of pp90 is an important determinant in the regulation of cellular proliferation by insulin.

The role of cAMP in regulating tumour cell growth.

Addition of cAMP to various cultured cell types has a dramatic effect on cell growth. Both positive and negative effects on growth have been demonstrated. Analysis of mutants with altered cAMP dependent protein kinases suggests that tumour cells do not require a functional endogenous cAMP system for normal cell cycling. Whether or not cAMP stimulates or inhibits cell growth depends on the cell type, the oncogene driving its growth, the dose of cAMP and the environment of the cell. The ras gene product does not appear to be a component of the adenylate cyclase system, and no other oncogenes have been shown to use cAMP as a second messenger. However, another class of oncogenes possesses a serine/threonine kinase activity analogous to that of cAMP dependent protein kinase. Several oncogene products and growth factor receptors are phosphorylated on serine residues, suggesting that some oncogene products, such as pp60src may be targets for the action of cAMP. The role of cAMP in tumour cell growth may be to modulate the activity of the oncogenes or their protein targets which control cell growth.

Strategies for whole microbial genome sequencing and analysis.

The introduction of methods for automated DNA sequence analysis nearly a decade ago, together with more recent advances in the field of bioinformatics, have revolutionized biology and medicine and have ushered in a new era of genomic science, the study of genes and genomes. These new technologies have had an impact on many areas of research, including the association between genes and disease, in DNA-based diagnostics, and in the sequencing of genomes from human and other model organisms. The demonstration in 1995, that automated DNA sequencing methods could be used to decipher the entire genome sequence of a free-living organism, Haemophilus influenzae, was a milestone in both the genomics and microbial fields [1]. Since the first report of the complete sequence of H. influenzae, these methodologies have been adopted by laboratories around the world. The complete genomic sequence of five eubacterial species [1-5], one archaea [6], and the eukaryote, Saccharomyces cerevisiae [7], have been reported in the last 18 months. At the beginning of 1997 more than a dozen microbial genome projects are at or near completion, with many others in progress. It is likely that in the next few years we will see the complete sequence of perhaps as many as 30-40 microbial genomes. In this article, we will review methods for whole genome sequencing and analysis and examine how this information can be exploited to better understand microbial physiology and evolution.

Ablation of stimulation of a cAMP-responsive promoter in CHO cell lines defective in their cAMP-dependent protein kinase system.

We have studied the requirement for an intact cAMP-dependent protein kinase (PKA) system to regulate cAMP-mediated gene transcription in Chinese hamster ovary (CHO) cells. Wild-type CHO cells and mutant CHO cell lines selected for their resistance to the growth inhibitory effect of 8-Br-cAMP and defective in their PKA system were transiently transfected with reporter plasmids containing 2.5 and 3.0 kb of the 5'-flanking sequence of the rat tyrosine aminotransferase (TAT) gene promoter. This segment of DNA contains no CRE-like sequences, yet wild-type transfectants exhibited a specific increase in TAT promoter activity following growth in medium containing 8-Br-cAMP. In CHO cell lines defective in their PKA, the transfected TAT promoter failed to respond to cAMP treatment. We conclude that an intact PKA system is necessary for the cAMP-mediated increase in TAT promoter activity in CHO cells and that there is no requirement for a CRE to see this effect.

The fine structure of the melanocytes of the adult mouse hair follicle during their amelanotic phase (telogen and early anagen).

Melanocyte-precursor cells have been described previously in telogen and early anagen hair germs by observation of Epon sections using light microscopy. The existence of these precursor cells has not been generally accepted due to lack of ultrastructural evidence. It is our purpose to describe these cells using transmission electron microscopy. We studied hair germs of the dorsal skin of adult mice in the appropriate stages of the hair cycle prior to melanogenesis. Telogen melanocytes are distinguishable from adjacent keratocytes by their lack of desmosomes, lack of coarse clumps of microfilaments, sparsity of ribosomes, unusually few nuclear pores and presence of dendrites. Melanocytes in early anagen show extensive growth with abundant polysomes, elaborate RER and Golgi zones. They still differ from keratocytes in their lack of desmosomes and clumps of microfilament, and also in the occasional presence of premelanosomes.

Comparative genomics and understanding of microbial biology.

The sequences of close to 30 microbial genomes have been completed during the past 5 years, and the sequences of more than 100 genomes should be completed in the next 2 to 4 years. Soon, completed microbial genome sequences will represent a collection of >200,000 predicted coding sequences. While analysis of a single genome provides tremendous biological insights on any given organism, comparative analysis of multiple genomes provides substantially more information on the physiology and evolution of microbial species and expands our ability to better assign putative function to predicted coding sequences.

Expression of Chinese hamster cAMP-dependent protein kinase in Escherichia coli results in growth inhibition of bacterial cells: a model system for the rapid screening of mutant type I regulatory subunits.

The regulatory and catalytic subunits of cAMP-dependent protein kinase (PKA) were coexpressed within the same bacterial cell using a polycistronic bacterial T7 expression vector encoding Chinese hamster cDNAs for the type I regulatory (RI) and catalytic alpha (C alpha) subunits of PKA. Basal expression of active RI/C alpha holoenzyme in the BL21(DE3) strain of Escherichia coli caused severe growth inhibition resulting in extremely small colony size. Several lines of evidence demonstrate that this growth inhibition requires active PKA subunits and cAMP: (i) this phenotype is dependent on cAMP since it is not seen in a strain lacking adenylyl cyclase activity, but the growth rate of these transformants is slower when exogenous cAMP is added; (ii) normal growth occurs when wild-type RI cDNA is replaced by a mutant RI cDNA encoding a RI protein with reduced cAMP binding; and (iii) the growth-inhibited phenotype of the transformed BL21(DE3) cells requires soluble, active C alpha protein. Holoenzyme expressed in bacteria is activated by cAMP, which stimulates phosphorylation of an endogenous 50-kDa protein that is missing in four host mutants selected for normal growth after transformation with PKA holoenzyme. A mutant RI cDNA library was generated by PCR random mutagenesis and screened by polycistronic expression in BL21(DE3) cells. The RI cDNA sequence from one revertant has base-pair substitutions creating two amino acid substitutions within the cAMP binding sites. The coexpression of the RI/C alpha subunits in BL21(DE3) bacterial cells provides a system for rapidly selecting mutations in the RI subunits of PKA.

Alignment of whole genomes.

A new system for aligning whole genome sequences is described. Using an efficient data structure called a suffix tree, the system is able to rapidly align sequences containing millions of nucleotides. Its use is demonstrated on two strains of Mycoplasma tuberculosis, on two less similar species of Mycoplasma bacteria and on two syntenic sequences from human chromosome 12 and mouse chromosome 6. In each case it found an alignment of the input sequences, using between 30 s and 2 min of computation time. From the system output, information on single nucleotide changes, translocations and homologous genes can easily be extracted. Use of the algorithm should facilitate analysis of syntenic chromosomal regions, strain-to-strain comparisons, evolutionary comparisons and genomic duplications.

Genomic loci of the Porphyromonas gingivalis insertion element IS1126.

The Porphyromonas gingivalis genome contains multiple copies of insertion element IS1126. When chromosomal DNA digests of different strains were probed with IS1126, between 25 and 35 hybridizing fragments per genome were detected, depending on the strain. Unrelated strains had very different restriction fragment length polymorphism (RFLP) patterns. When different laboratory copies of a specific strain were examined, the IS1126 RFLP patterns were very similar but small differences were observed, indicating that element-associated changes had occurred during laboratory passage. Within the next year, genome sequencing, assembly, and annotation for P. gingivalis W83 will be completed. Because repetitive elements complicate the assembly of randomly sequenced DNA fragments, we isolated and sequenced the flanking regions of IS1126 copies in strain W83. We also isolated and sequenced the flanking regions of IS1126 copies in strain ATCC 33277 in order to compare insertion sites in phylogenetically divergent strains. We identified 37 new sequences flanking IS1126 from strain ATCC 33277 and 30 from strain W83. The insertion element was found between genes except where it transposed into another insertion element. Examination of identifiable flanking genes or open reading frames indicated that the insertion sites were different in the two strains, except that both strains possess an insertion adjacent to the Lys-gingipain gene (J. P. Lewis and F. L. Macrina, Infect. Immun. 66:3035-3042, 1998). Most of the genes or sequences flanking IS1126 in ATCC 33277 were present in W83 but were contiguous and not insertion element associated. Thus, where genes were identified in both strains, their order was maintained, indicating that the two genomes are organized similarly, but the loci of IS1126 are different. In both strains, insertion element-associated duplicated target sites were lost from several copies of IS1126, providing evidence of homologous recombination between elements. Larger organizational differences between the genomes, such as deletions and inversions, may result from insertion element-mediated recombination events.