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Many proteins synthesized through the secretory pathway receive posttranslational modifications, including N-glycosylation. [alpha]-Mannosidase II (MII) is a key enzyme converting precursor high-mannose-type N-glycans to matured complex-type structures. Previous studies showed that MII-null mice synthesize complextype N-glycans, indicating the presence of an alternative pathway. Because [alpha]-mannosidase IIx (MX) is a candidate enzyme for this pathway, we asked whether MX functions in N-glycan processing by generating MII/MX double-null mice. Some double-nulls died between embryonic days 15.5 and 18.5, but most survived until shortly after birth and died of respiratory failure, which represents a more severe phenotype than that seen in single-nulls for either gene. Structural analysis of N-glycans revealed that double-nulls completely lack complex-type N-glycans, demonstrating a critical role for at least one of these enzymes for effective N-glycan processing. Recombinant mouse MX and MII showed identical substrate specificities toward N-glycan substrates, suggesting that MX is an isozyme of MII. Thus, either MII or MX can biochemically compensate for the deficiency of the other in vivo, and either of two is required for late embryonic and early postnatal development. geneknockout | mutation

To understand the complicated interplay when a traveling myosin head reaches interaction distance with two actins in a filament we looked to three myosin loops that early on exert their influences from the 'outside' of the myosin. On these we conduct, functionally test, and interpret strategically chosen mutations at sites thought from crystallography to be a patch for binding the 'first' of the two actins. One loop bears a hydrophobic triplet of residues, one is the so-called 'loop 2,' and the third is the 'cardiomyopathy' loop. So far as we know, the myosin sites that first respond are the two lysine-rich loops that produce an ionic strength-dependent weak-binding complex with actin. Subsequently, the three loops of interest bind the first actin simultaneously, and all three assist in closing the cleft in the 50-kDa domain of the myosin, a closure that results in transition from weak to strong binding and precedes rapid [P.sub.i] release and motility. Mutational analysis shows that each such loop contact is distinctive in the route by which it communicates with its specific target elsewhere in myosin. The strongest contact with actin, for example, is that of the triplet-bearing loop. On the other hand, that of loop 2 (dependent on drawing close two myosin lysines and two actin aspartates) is probably responsible for opening switch I and uncovering the [gamma]-phosphate moiety of bound ATP. Taking into account these findings, we begin to arrange in order many molecular events in muscle function. site-directed mutation | hydrophobic triplet | loop 2 | cardiomyopathy loop | pyrene-actin fluorescence

Water-specific aquaporins (AQP), such as the prototypical mammalian AQP1, stringently exclude the passage of solutes, ions, and even protons. Supposedly, this is accomplished by two conserved regions within the pore, a pair of canonical asparagine-proline-alanine (NPA) motifs, the central constriction, and an aromatic/ arginine (ar/R) constriction, the outer constriction. Here, we analyzed the function of three residues in the ar/R constriction (Phe-56, His-180, and Arg-195) in rat AQP1. Individual or joint replacement of His-180 and Arg-195 by alanine and valine residues, respectively (AQP1-H180A, AQP1-R195V, and AQP1-H180A/ R195V), did not affect water permeability. The double mutant AQP1-H180A/R195V allowed urea to pass. In line with the predicted solute discrimination by size, replacement of both Phe-56 and His-180 (AQP1-F56A/H180A) enlarged the maximal diameter of the ar/R constriction 3-fold and enabled glycerol and urea to pass. We further show that ammonia passes through all four AQP1 mutants, as determined (i) by growth complementation of yeast deletion strains with ammonia, (ii) by ammonia uptake from the external solution into oocytes, and (iii) by direct recordings of ammonia induced proton currents in oocytes. Unexpectedly, removal of the positive charge in the ar/R constriction in AQP1-R195V and AQP1-H180A/R195V appeared to allow the passage of protons through AQP1. The data indicate that the ar/R constriction is a major checkpoint for solute permeability, and that the exquisite electrostatic proton barrier in AQPs comprises both the NPA constriction as well as the ar/R constriction. mutational analysis | proton filter | solute selectivity

Frame-shift mutations at microsatellites occur as a time-dependent function of polymerase errors followed by failure of postreplicational mismatch repair. A cell-culture system was developed that allows identification of intermediate mutant cells that carry the mutation on a single DNA strand after the initial DNA polymerase errors. A plasmid was constructed that contained 13 repeats of a poly(dC-dA).poly(dG-dT) oligonucleotide immediately after the translation initiation codon of the enhanced GFP (EGFP) gene, shifting the EGFP gene out of its proper reading frame. The plasmid was introduced into human mismatch repair-deficient (HCT116, hMLH1-mutated) and mismatch repair-proficient (HCT116+chr3, hMLH1 wild type) colorectal cancer cells. After frame-shift mutations occurred that restored the EGFP reading frame, EGFP-expressing cells were detected, and two distinct fluorescent populations, M1 (dim cells) and M2 (bright cells), were identified. M1 cell numbers were stable, whereas M2 cells accumulated over time. In HCT116, single M2 cells gave rise to fluorescent colonies that carried a 2-bp deletion at the [(CA).sub.13] microsatellite, Twenty-eight percent of single M1 cells, however, gave rise to colonies with a mixed fluorescence pattern that carried both [(CA).sup.13] and [(CA).sub.12] microsatellites. It is likely that M1 cells represent intermediate mutants that carry [(CA).sub.13]*[(GT).sub.12] heteroduplexes. Although the mutation rate in HCT116 cell clones (6.2 x [10.sup.-4]) was 30 times higher than in HCT116+chr3 (1.9 x [10.sup.-5]), the proportion of M1 cells in culture did not significantly differ between HCT116 (5.87 x [10.sup.-3]) and HCT116+chr3 (4.13 x [10.sup.-3]), indicating that the generation of intermediate mutants is not affected by mismatch-repair proficiency. mismatch repair | frame-shift mutation | microsatellite instability | mutation rate

Although vaccine-induced immunity to influenza A virus is continually challenged by progressively selected mutations in the virus's major antigens (antigenic drift), virus strains within a subtype (e.g., H1N1) are antigenically cross-reactive. Although cross-immunity diminishes as further mutations accumulate, necessitating frequent changes in vaccine strains, older vaccines are usually partially protective. The post-World War II epidemic of 1947 is notable for the total failure of a vaccine previously effective in the 1943-44 and 1944-45 seasons. We have combined extensive antigenic characterization of the hemagglutinin and neuraminidase antigens of the 1943 and 1947 viruses with analysis of their nucleotide and amino acid sequences and have found marked antigenic and amino acid differences in viruses of the two years. Furthermore, in a mouse model, vaccination with the 1943 vaccine had no effect on infection with the 1947 strain. These findings are important, because complete lack of cross-immunogenicity has been found previously only with antigenic shift, in which antigenically novel antigens have been captured by reassortment of human and animal strains, sometimes leading to pandemics. Although the 1947 epidemic lacked the usual hallmarks of pandemic disease, including an extensive increase in mortality, it warns of the possibility that extreme intrasubtypic antigenic variation (if coupled with an increase in disease severity) could produce pandemic disease without the introduction of animal virus antigens.

Vlad tepes ([vlt.sup.m651]) is one of only five 'bloodless' zebrafish mutants isolated through large-scale chemical mutagenesis screening. It is characterized by a severe reduction in blood cell progenitors and few or no blood cells at the onset of circulation. We now report characterization of the mutant phenotype and the identification of the gene mutated in [vlt.sup.m651]. Embryos homozygous for the [vlt.sup.m651] mutation had normal expression of hematopoietic stem cell markers through 24 h postfertilization, as well as normal expression of myeloid and lymphoid markers. Analysis of erythroid development revealed variable expression of erythroid markers. Through positional and candidate gene cloning approaches we identified a nonsense mutation in the gata1 gene, 1015C [right arrow] T (Arg-339 [right arrow] Stop), in [vlt.sup.m651]. The nonsense mutation was located C-terminal to the two zinc fingers and resulted in a truncated protein that was unable to bind DNA or mediate GATA-specific transactivation. A BAC clone containing the zebrafish gata1 gene was able to rescue the bloodless phenotype in [vlt.sup.m651]. These results show that the [vlt.sup.m651] mutation is a previously uncharacterized gata1 allele in the zebrafish. The [vlt.sup.m651] mutation sheds new light on Gata1 structure and function in vivo, demonstrates that Gata1 plays an essential role in zebrafish hematopoiesis with significant conservation of function between mammals and zebrafish, and offers a powerful tool for future studies of the hematopoietic pathway.

The transthyretin (TTR) amyloid diseases are of keen interest, because there are >80 mutations that cause, and a few mutations that suppress, disease. The V122I variant is the most common amyloidogenic mutation worldwide, producing familial amyloidotic cardiomyopathy primarily in individuals of African descent. The substitution shifts the tetramer-folded monomer equilibrium toward monomer (lowers tetramer stability) and lowers the kinetic barrier associated with rate-limiting tetramer dissociation (pH 7; relative to wild-type TTR) required for amyloid fibril formation. Fibril formation is also accelerated because the folded monomer resulting from the tetramer-folded monomer equilibrium rapidly undergoes partial denaturation and self-assembles into amyloid (in vitro) when subjected to a mild denaturation stress (e.g., pH 4.8). Incorporation of the V122I mutation into a folded monomeric variant of transthyretin reveals that this mutation does not destabilize the tertiary structure or alter the rate of amyloidogenesis relative to the wild-type monomer. The increase in the velocity of rate-limiting tetramer dissociation coupled with the lowered tetramer stability (increasing the mol fraction of folded monomer present at equilibrium) may explain why V122I confers an apparent absolute anatomic risk for cardiac amyloid deposition.

The PU.1 interaction partner (Pip) is a member of the interferon regulatory factor family that regulates gene expression through heterodimerization with the ETS transcription factor PU.1. Binding of Pip alone to DNA is weak, and usually it is recruited by phosphorylated PU.1 to form a strong ternary complex with specific DNA sequences. An approach combining sequence homology analysis, secondary structure predictions, and a precise mutational strategy has been used to determine critical residues within the Pip heterodimerization domain that contribute to ternary complex formation. We have delimited the Pip interaction domain to residues 245-422 by using deletion analysis. Site-directed mutagenesis of conserved polar amino acids within two predicted [Alpha]-helices contained in this region, and which are highly conserved in the IRF family, confirmed the importance of these residues for Pip-PU.1 interaction with DNA as well as for trans-activation activity. Our results suggest the existence of a functional epitope essential for heterodimerization between Pip and PU.1 and possibly, in general, between interferon regulatory factor family members and their partners.

The spectrum of mutations induced by the naturally occurring DNA adduct pyrimido[1,2-[Alpha]]purin-10(3H)-one ([M.sub.1]G) was determined by site-specific approaches using M13 vectors replicated in Escherichia coli. [M.sub.1]G was placed at position 6256 in the (-)-strand of M13MB102 by ligating the oligodeoxynucleotide 5[prime]-GGT([M.sub.1]G)TCCG-3[prime] into a gapped-duplex derivative of the vector. Unmodified and [M.sub.1]G-modified genomes containing either a cytosine or thy-mine at position 6256 of the (+)-strand were transformed into repair-proficient and repair-deficient E. coli strains, and base pair substitutions were quantitated by hybridization analysis. Modified genomes containing a cytosine opposite [M.sub.1]G resulted in roughly equal numbers of [M.sub.1]G[approaches]A and [M.sub.1]G[approaches]T mutations with few [M.sub.1]G[approaches]C mutations. The total mutation frequency was [approximately equal to]1%, which represents a 500-fold increase in mutations compared with unmodified M13MB102. Transformation of modified genomes containing a thymine opposite [M.sub.1]G allowed an estimate to be made of the ability of [M.sub.1]G to block replication. The (-)-strand was replicated >80% of the time in the unadducted genome but only 20% of the time when [M.sub.1]G was present. Correction of the mutation frequency for the strand bias of replication indicated that the actual frequency of mutations induced by [M.sub.1]G was 18%. Experiments using E. coli with different genetic backgrounds indicated that the SOS response enhances the mutagenicity of [M.sub.1]G and that [M.sub.1]G is a substrate for repair by the nucleotide excision repair complex. These studies indicate that [M.sub.1]G, which is present endogenously in DNA of healthy human beings, is a strong block to replication and an efficient premutagenic lesion.

The xeroderma pigmentosum group D (XPD) protein has a dual function, both in nucleotide excision repair of DNA damage and in basal transcription. Mutations in the XPD gene can result in three distinct clinical pheno-types, XP, trichothiodystrophy (TTD), and XP with Cockayne syndrome. To determine if the clinical phenotypes of XP and TTD can be attributed to the sites of the mutations, we have identified the mutations in a large group of TTD and XP-D patients. Most sites of mutations differed between XP and TTD, but there are three sites at which the same mutation is found in XP and TTD patients. Since the corresponding patients were all compound heterozygotes with different mutations in the two alleles, the alleles were tested separately in a yeast complementation assay. The mutations which are found in both XP and TTD patients behaved as null alleles, suggesting that the disease phenotype was determined by the other allele. If we eliminate the null mutations, the remaining mutagenic pattern is consistent with the site of the mutation determining the phenotype.

We present a further development in the technology of sequencing by hybridization to oligonucleotide microchips (SHOM) and its application to diagnostics for genetic diseases. A robot has been constructed to manufacture sequencing 'microchips.' The microchip is an array of oligonucleotides immobilized into gel elements fixed on a glass plate. Hybridization of the microchip with fluorescently labeled DNA was monitored in real time simultaneously for all microchip elements with a two-wavelength fluorescent microscope equipped with a charge-coupled device camera. SHOM has been used to detect [Beta]-thalassemia mutations in patients by hybridizing PCR-amplified DNA with the microchips. A contiguous stacking hybridization technique has been applied for the detection of mutations; it can simplify medical diagnostics and enhance its reliability. The use of multicolor monitoring of contiguous stacking hybridization is suggested for large-scale diagnostics and gene polymorphism studies. Other applications of the SHOM technology are discussed.

L125R is a mutation in the transmembrane helix C of rhodopsin that is associated with autosomal dominant retinitis pigmentosa. To probe the orientation of the helix and its packing in the transmembrane domain, we have prepared and studied the mutations E122R, I123R, A124R, S127R, L125F, and L125A at, and in proximity to, the above mutation site. Like L125R, the opsin expressed in COS-1 cells from E122R did not bind 11-cis-retinal, whereas those from I123R and S127R formed the rhodopsin chromophore partially. A124R opsin formed the rhodopsin chromophore ([[Lambda].sub.max] 495 nm) in the dark, but the metarhodopsin II formed on illumination decayed about 6.5 times faster than that of the wild type and was defective in transducin activation. The mutant opsins from L125F and L125A bound 11-cis-retinal only partially, and in both cases, the mixtures of the proteins produced were separated into retinal-binding and non-retinal-binding (misfolded) fractions. The purified mutant rhodopsin from L125F showed [[Lambda].sub.max] at 500 nm, whereas that from L125A showed [[Lambda].sub.max] at 503 nm. The mutant rhodopsin L125F showed abnormal bleaching behavior and both mutants on illumination showed destabilized metarhodopsin II species and reduced transducin activation. Because previous results have indicated that misfolding in rhodopsin is due to the formation of a disulfide bond other than the normal disulfide bond between Cys-110 and Cys-187 in the intradiscal domain, we conclude from the misfolding in mutants L125F and L125A that the folding in vivo in the transmembrane domain is coupled to that in the intradiscal domain.

The rhodopsin mutants P23H and G188R, identified in autosomal dominant retinitis pigmentosa (ADRP), and the site-specific mutants D190A and [Delta]Y191-Y192 were expressed in COS cells from synthetic mutant opsin genes containing these mutations. The proteins expressed from P23H and D190A partially regenerated the rhodopsin chromophore with 11-cis-retinal and were mixtures of the correctly folded (retinal-binding) and misfolded (non-retinal-binding) opsins. The mixtures were separated into pure, correctly folded mutant rhodopsins and misfolded opsins. The proteins expressed from the ADRP mutant G188R and the mutant [Delta]Y191-Y192 were composed of totally misfolded non-retinal-binding opsins. Far-UV CD spectra showed that the correctly folded mutant rhodopsins had helical content similar to that of the wild-type rhodopsin, whereas the misfolded opsins had helical content 50-70% of the wild type. The near-UV CD spectra of the misfolded mutant proteins lack the characteristic band pattern seen in the wild-type opsin, indicative of a different tertiary structure. Further, whereas the folded mutant rhodopsins were essentially resistant to trypsin digestion, the misfolded opsins were degraded to small fragments under the same conditions. Therefore, the misfolded opsins appear to be less compact in their structures than the correctly folded forms. We suggest that most, if not all, of the point mutations in the intradiscal domain identified in ADRP cause partial or complete misfolding of rhodopsin.

Recombination repair protein 1 Rrp1 includes a C-terminal region homologous to several DNA repair proteins, including Escherichia cold exonuclease III and human APE, that repair oxidative and alkylation damage to DNA. The nuclease activities of Rrp1 include apurinic/apyrimidinic endonuclease, 3'-phosphodiesterase, 3'-pbosphatase, and 3'-exonuclease. As shown previously, the C-terminal nuclease region of Rrp1 is sufficient to repair oxidative- and alkylation-induced DNA damage in repair-deficient E. cold mutants. DNA strand-transfer and single-stranded DNA renaturation activities are associated with the unique N-terminal region of Rrp1, which suggests possible additional functions that include recombinational repair or homologous recombination. By using the Drosophila w/[w.sup.+] mosaic eye system, which detects loss of heterozygosity as changes in eye pigmentation, somatic mutation and recombination frequencies were determined in transgenic flies overexpressing wild-type Rrp1 protein from a heat-shock-inducible transgene. A large decrease in mosaic clone freguency is observed when Rrp1 overexpression precedes treatment with [gamma]-rays, bleomycin, or paraquat. In contrast, Rrp1 overexpression does not alter the spot frequency after treatment with the alkylating agents methyl methanesulfonate or methyl nitrosourea. A reduction in mosaic clone frequency depends on the expression of the Rrp1 transgene and on the nature of the induced DNA damage. These data suggest a lesion-specific involvement of Rrp1 in the repair of oxidative DNA damage.

Long CTG triplet repeats which are associated with several human hereditary neuromuscular disease genes are stabilized in ColE1-derived plasmids in Escherichia coli containing mutations in the methyl-directed mismatch repair genes (mutS, mutL, or mutH). When plasmids containing (CTG[).sub.180] were grown for about 100 generations in muts, mutl, or muth strains, 60-85% of the plasmids contained a full-length repeat, whereas in the parent strain only about 20% of the plasmids contained the full-length repeat. The deletions occur only in the (CTG[).sub.180] insert, not in DNA flanking the repeat. While many products of the deletions are heterogeneous in length, preferential deletion products of about 140, 100, 60, and 20 repeats were observed. We propose that the E. coli mismatch repair proteins recoginize three-base loops formed during replication and then generate long singlestranded gaps where stable hairpin structures may form which can be bypassed by DNA polymerase during the resynthesis of duplex DNA. Similar studies were conducted with plasmids containing CGG repeats; no stabilization of these triplets was found in the mismatch repair mutants. Since prokaryotic and human mismatch repair proteins are similar, and since several carcinoma cell lines which are defective in mismatch repair show instability of simple DNA microsatellites, these mechanistic investigations in a bacterial cell may provide insights into the molecular basis for some human genetic diseases.

Malignant mesotheliomas (MMs) are aggressive tumors that develop most frequently in the pleura of patients exposed to asbestos. In contrast to many other cancers, relatively few molecular alterations have been described in MMs. The most frequent numerical cytogenetic abnormality in MMs is loss of chromosome 22. The neurofibromatosis type 2 gene (NF2) is a tumor suppressor gene assigned to chromosome 22q which plays an important role in the development of familial and spontaneous tumors of neuroectodermal origin. Although MMs have a different histogenic derivation, the frequent abnormalities of chromosome 22 warranted an investigation of the NF2 gene in these tumors. Both cDNAs from t5 MM cell lines and genomic DNAs from 7 matched primary tumors were analyzed for mutations within the NF2 coding region. NF2 mutations predicting either interstitial in-frame deletions or truncation of the NF2-encoded protein (merlin) were detected in eight cell lines (53%), six of which were confirmed in primary tumor DNAs. In two samples that showed NF2 gene transcript alterations, no genomic DNA mutations were detected, suggesting that aberrant splicing may constitute an additional mechanism for merlin inactivation. These findings implicate NF2 in the oncogenesis of primary MMs and provide evidence that this gene can be involved in the development of tumors other than nervous system neoplasms characteristic of the NF2 disorder. In addition, unlike NF2-related tumors, MM derives from the mesoderm; malignancies of this origin have not previously been associated with frequent alterations of the NF2 gene.

Eukaryotic genomes contain tracts of DNA in which a single base or a small number of bases are repeated (microsatellites). Mutations in the yeast DNA mismatch repair genes MSH2, PMS1, and MLH1 increase the frequency of mutations for normal DNA sequences and destabilize microsatellites. Mutations of human homologs of MSH2, PMSI, and MLH1 also cause microsatellite instability and result in certain types of cancer. We find that a mutation in the yeast gene MSH3 that does not substantially affect the rate of spontaneous mutations at several loci increases microsatellite instability about 40-fold, preferentially causing deletions. We suggest that MSH3 has different substrate specificities than the other mismatch repair proteins and that the human MSH3 homolog (MRP1) may be mutated in some tumors with microsatellite instability.

Mouse skin tumors contain activated c-H-ras oncogenes, often caused by point mutations at codons 12 and 13 in exon 1 and codons 59 and 61 in exon 2. Mutagenesis by the noncoding apurinic sites can produce G [right arrow] T and A [right arrow] T transversions by DNA misreplication with more frequent insertion of deuxyadenosine opposite the apurinic site. Papillomas were induced in mouse skin by several aromatic hydrocarbons, and mutations in the c-H-ras gene were determined to elucidate the relationship among DNA adducts, apurinic sites, and ras oncogene mutations. Dibenzo-[a,l]pyrene (DB[a,l]P), DB[a,l]P-11,12-dihydrodiol, anti-DB[a,l]P-11,12-diol-13,14-epoxide, DB[a,l]P-8,9-dihydrodiol, 7,12-dimethylbenz[a]anthracene (DMBA), and 1,2,3,4-tetrahydro-DMBA consistently induced a CAA [right arrow] CTA mutation in codon 61 of the c-H-ras oncogene. Benzo[a]pyrene induced a GGC [right arrow] GTC mutation in codon 13 in 54% of tumors and a CAA [right arrow] CTA mutation in codon 61 in 15%. The pattern of mutations induced by each hydrocarbon correlated with its profile of DNA adducts. For example, both DB[a,l]P and DMBA primarily form DNA adducts at the N-3 and/or N-7 of deoxyadenosine that are lost from the DNA by depurination, generating apurinic sites. Thus, these results support the hypothesis that misreplication of unrepaired apurinic sites generated by loss of hydrocarbon-DNA adducts is responsible for transforming mutations leading to papillomas in mouse skin.

Production of infectious human immunodeficiency virus (HIV) requires proper polyprotein processing by the dimeric viral protease. The trans-dominant inhibitory activity of a defective protease monomer with the active site Asp-25 changed to Asn was measured by transient transfection. A proviral plasmid that included the drug-selectable Escherichia coligpt gene was used to deliver the wild-type (wt) or mutant proteases to cultured-cells. Coexpression of the wt proviral DNA (HIV-gpt) with increasing amounts of the mutant proviral DNA (HIY-gpt D25N) results in a concomitant decrease in proteolytic activity monitored by in vivo viral polyprotein processing. The viral particles resulting from inactivation of the protease were mostly immature, consisting predominantly of unprocessed [p55.sup.gag] and [pl60.sup.gag-pol] polyproteins. In the presence of HIV-1 gpl60 env, the number of secreted noninfectious particles correlated with the presence of increasing amounts of the defective protease. Greater than 97% reduction in infectivity was observed at a 1:6 ratio of wt to defective protease DNA. This provides an estimate of the level of inhibition required for effectively preventing virion processing. Stable expression of the defective protease in monkey cells reduced the yield of infectious particles from these cells by 90% upon transfection with the wt proviral DNA. These results show that defective subunits of the viral protease exert a trans-dominant inhibitory effect resulting from the formation of catalytically compromised heterodimers in vivo, ultimately yielding noninfectious viral particles.

The block model of protein suggests that the mutation in one block affects the mutation of another independent block. These blocks are represented by the amino acids in the tertiary structure domains. Short-range correlations are introduced among neighbouring blocks to make mutational changes the same at a particular point and at a definite time. The number of blocks decides the amount of correlation among them. This block theory can be applied to the somatic hypermutation of the antibody molecules.

The identification of the mutations in genes for transcription factors, cell adhesion molecules, intercellular junctions, receptors, molecules used in signal transduction, growth factors, structural enzymes, proteins and transporters is important for identifying the birth defects. On analyzing the developmental influences, the presence of natural or engineered models in the mouse and in Drosophila are helpful.

Mutational analysis of phytochrome B (phyB) identifies a small COOH-terminal-domain region critical for regulatory activity of both phyB and phyA. The mutant molecules work during photosignal perception but do not work during the signal transfer to downstream components. All four mutations isolated by the mutagenesis of transgenic phyB-overexpressing Arabidopsis are contained within a 62-residue region of phyB's COOH-terminal domain.

Alzheimer's disease (AD) is genetically heterogeneous and developing AD epsilon-4 allele is a risk factor. Early-onset autosomal dominant AD originates from the mutations in the amyloid precursor protein gene (APP), but this constitute only 5% or less of cases. According to in vitro studies the cells expressing mutant APPs in large quantities generate pathogenic A-beta peptide which is the main factor in the AD amyloid.

A Mendelian form of hypertension called glucocorticoid-remediable aldosteronism occurs due to mutations causing ectopic expression of aldosterone synthase enzymatic activity. Liddle syndrome, the Mendelian form of hypertension is activated due to the mutations in the beta subunit of the amiloride-sensitive epithelial sodium channel. The general variants at the angiotensinogen locus are responsible for the pathogenesis of the required hypertension in Caucasian subjects.

Four different transposable elements are mobilized in a system of hybrid dysgenesis in Drosophila virilis in crosses between females of strain B9 and males of strain L160. The F1 progeny of the dysgenic cross shows gonadal dystrophy, a high degree of sterility and new mutations, male recombination, chromosome nondisjunction, and transmission ratio distortion. Consistency of the results with the McClintock's model is discussed.

The etiology of schizophrenia is thought to be both genetic and experiential. A genetic study of a group of schizophrenic patients shows two interstitial deletions on chromosome 22q11. The size of the deletions is calculated to be between 1.5 to 2 megabases. Another purpose of the study was to see if differences in deletion size were related to the schizophrenic phenotype in patients with velocardiofacial syndrome. Details of study on overlap between schizophrenia and velocardiofacial syndrome and the results are described.

VHL tumor suppressor gene, a nuclear protein implicated in renal carcinoma and von Hippel-Lindau disease can translocate between the nucleus and the cytosol and executes its function by formation of specific multiprotein complexes. Both human and analogus rat VHL proteins are conserved in subcellular localisation and also in their ability to form protein complexes. VHL plays a regulatory role in nuclear events related to progression of cell cycle and gene transcription. Loss of specific complex formation in naturally occurring inactivating mutants of VHL is demonstrated.

Camptothecin, a potent antineoplastic agent reversibly stabilizes a covalent enzyme-DNA intermediate formed by DNA topoisomerase I, and leads to G(sub 2) arrest and cell mortality. When ethyl methanesulfonate mutagenesis was carried out on yeast cells expressing plasmid borne wild-type DNA topoisomerase I, six dominant suppressors of camptothecin toxicity were shown to comprise a single genetic locus, SCT1. SCT1 cell sensitivity to other DNA-damaging agents suggests that changes in SCT1 function inhibit camptothecin-induced DNA damage caused in the vicinity of yeast DNA topoisomerase I.

Common catalytic mechanisms are shared by PTPases and dual specific phosphatases. Cysteine is probably the active site nucleophile which can form a thiol-phosphate intermediate. The mutation of serine-131 to an alanine (S131A) in the dual specific protein-tyrosine phosphatase called VHR changes the rate limiting step in the catalytic mechanism by formation of a rate-limiting phosphoenzyme. Researchers observed a burst of p-nitrophenol formation appears when p-nitrophenol phosphate reacts with S131 enzyme. Loss of the hydroxyl group at the active site reduces the ability to hydrolyze the thiol-phosphate intermediate.

In human bone marrow cells, benzene develops gene-duplicating mutations at the glycophorin A (GPA) locus. Enhanced exposure to benzene increases the cell frequency of the surface protein with double N expression (NN), but not of the surface protein with single N. Duplication of the N allele and loss of the GPA M allele produces the NN variant. The surface protein with single N allele results from gene inactivation.

The mouse Splotch-delayed (super d) mutation suggests the dependency of paired domain and homeodomain of Pax-3 protein on each other and an impairment in the DNA-binding properties of Pax-3. An analysis of the DNA-binding properties of wild-type and Sp(super d) Pax-3 indicates that the wild-type binds e5 in a particular pattern whereas the Sp(super d) mutation reduces binding of Pax-3 to e5 by seventeen times.

Studies using eight lines of transgenic mice produced data inconsistent with current hypotheses regarding rhodopsin mutations causing autosomal dominant retinitis pigmentosa. K296E in the rhodopsin gene produces an opsin which was not activated by phosphorylation or arrestin, but is permanently bound to arrestin. Therefore, K296E opsin phototransduction constitutive activation does not cause photoreceptor death.

The suppressors of a transcription factor TFIIB defect, obtained from mutations in the Saccharomyces cerevisiae SSU71 gene, provide a cold-sensitive growth defect and a downstream shift in transcription start-site selection at the cyc1 locus. The conditional phenotype is restricted and the transcriptional initiation design is preserved by the ssu71-1 suppressor. SSU71 is a single-copy gene that encodes a highly charged protein having a molecular mass of 82194 daltons. It is similar to subunits of the transcription factor TFIIF.

Mutations in the polymerase domain of reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) confers resistance to multiple antiretroviral dideoxynucleosides on HIV-1 variants in patients undergoing chemotherapy with dideoxynucleosides. Mutation at codon 151 is crucial for the development of resistance and mutations of codons 62, 75, 77, 106 and 151 occur at sites proximal to the dNTP-binding site of RT and the first nucleotide of the single-stranded template.

Ribosomal frameshift provides partial rescue of the human carbonic anhydrase II frameshift mutation. Normal reading frame is restored by the frameshift after introducing 11 missense amino acids and the frameshift also helps complete translation in the normal reading frame. An antibody is produced against the 11-amino acid frameshift peptide and the 29-kDa double-frameshift protein is generated in COS cells with the Hispanic mutant cDNA.

Analysis of genomic DNA from 32 unrelated families with male-limit precocious puberty subjected to Asp578 to Gly, Met571 to Ile and Thr577 to Ile mutations in transmembrane helix 6 of the human luteinizing hormone receptor (hLHR) shows that heterogeneous point mutations occur mainly in the region covering nt 1624-1741 of exon 11. The mutations trigger hLHR to result in male-limited precocious puberty.

A study of the germinal revertant of an Adh-1-3F 1124 mutation of the maize alcohol dehydrogenase 1 gene adh by introducing the transposon Mu 3 at the TATA box of the promoter reveals that a complex, multibreak-point DNA rearrangement with deletion, inverted duplication and inversions varying 430 nucleotides in the promoter region arises due to the excision of Mu 3. adh1 gene expression pattern is altered, with one organ exhibiting high enzyme levels, another exhibiting decreased levels, while a third organ maintains enzyme activity levels. A fragmentation model is proposed for the stimulation of multiple breakpoint aberrations by transposon excision, without the involvement of a homologous chromosome.

A mutant (LTK7) of the Escherichia coli heat-labile enterotoxin (LT) that is deficient in ADP-ribosylation activity but forms holotoxin, is highly nontoxic and serves as an effective intranasal mucosal adjuvant. Mutation at the arginine at site 7 allows the molecules to bind to eukaryotic cells and to serve as a mucosal adjuvant for co-treated proteins. Intranasal immunization with LTK7 and ovalbumin increases the levels of serum and local antibodies to ovalbumin and toxin.

The biology and biochemistry of epidermal growth factor receptors (EGFR) undergo variations due to the presence of a point mutation waved-2 (wa-2) in the gene encoding EGFR. This mutation, which causes a valine to glycine substitution at residue 743, affects lactation in mothers and leads to death of the offsprings. The young or adult w-2 mice remain unaffected by defective EGFR signaling.

An study of the acetylcholine receptor (AChR) subunit genes through molecular genetic analysis indicates the prolonged openings in the AChR channel to be due to disruption of the putative M2 alpha-helix, caused by a Thr-264 to Pro mutation. This mutation is present in the nucleotide 790 in exon 8 of the epsilon subunit gene. This variation in the kinetics of the AChR channels leads to congenital myasthenic syndrome, as suggested by patch-clamp studies.

A study of mice obtained from embryonic stem (ES) cells mutated at the interleukin 2 receptor gamma chain (IL-2Rgamma) by Cre loxP-mediated recombination shows that IL-2Rgamma mediates the production and activity of mainstream B- and T-cells and also contributes to natural killer (NK)-cell development. The mutant mice deficient in IL-2R gamma show reduced levels of B, T and NK cells. Mutation also attenuates gut-associated intraepithelial lymphocytes and abolishes the Peyer's patches.

Analysis of four probable mismatch sets possessing 8 single-base-pair mismatches obtained from isolated mutations helped assess the potential of T4 endonuclease VII to identify heteroduplex mismatches. Cleavage occurs in at least one strand of each set that contains a mismatch while an A.A T.T set remains uncleaved in any mismatched strand. This technique facilitates the efficient detection and location of all mutational changes upon DNA screening for mutations.

Expression of six skeletal muscle voltage-gated Na+ channel alpha-subunit mutations from patients with paramyotonia congenita (PC) and hyperkalemic periodic paralysis (HYPP) in a heterologous cell line (tsA201 cells) indicates that all the PC mutants display similar channel inactivation properties. One HYPP mutation displays normal inactivation rate with changes in the midpoints of inactivation and activation in the voltage axis. The lack of any defect in the activation by PC mutants indicates the phenotypic variations between the biophysical and molecular effects of PC and HYPP mutants.

Analysis of the structural framework responsible for the chromosome X-linked agammaglobulinemia (XLA) disease in man using a three-dimensional model for the Bruton tyrosine kinase (BTK) kinase domain reveals that XLA is a tyrosine kinase disease. The Arg-525 to Gln mutation nullifies the tyrosine kinase activity of the BTK domain. The specific localization of the mutations isolated from XLA patients to one side of the BTK kinase domain indicates the structural grouping of functionally vital residues.

Targeting of the tsA58 mutation of simian virus 40 large tumor antigen to megakaryocytes results in the disturbance of megakaryocyte differentiation by the T antigen in the transgenic mice. Low platelets levels and large tumor antigen protein levels were seen in the affected cells. The morphological characteristics of the megakaryocytes and platelets in the thrombocytopenic mice are abnormal. A genetic model for megakaryocytic neoplasia and thrombocytopenia in the mice indicates the involvement of the retinoblastoma protein in megakaryocyte differentiation.

Tagging of the Tox1 locus in the filamentous Ascomycete Cochliobolus heterostrophus by an insertional mutagenesis procedure proceeds through restriction enzyme-mediated integration. Tox1 tagging involves the transformation of a Tox1+ strain with a linearized, nonhomologous plasmid and an excess of restriction enzyme to linearize the plasmid. The Tox1 locus is a single genetic element that regulates the formation of the linear polyketide T toxin which is a fungal virulence factor.

Analysis of somatic mutations of mouse immunoglobulin lambda1 light chains from Peyer's patches germinal center B cells reveals that the hypermutation rates of endogenous lambda 1 genes agree with that of a kappa transgene . Both in-frame and out-of-frame rearrangements have common intrinsic hot spots of mutation that cluster in hypermutating domains. These hot spot cluster in complementarity-determining region (CDR)1, CDR2 and CDR3 and the mutations also cluster in the joining/constant region intron.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria derives its visible absorbance and fluorescence from a p-hydroxybenzylidene-imidazolidinone chromophore that is obtained upon cyclization and autoxidation of its Ser-Tyr-Gly sequence at residues 65-67. Mutagenic studies reveal that the final fluoro-formation requires molecular oxygen and progresses via a dilution-independent time constant. GFP can be utilized in molecular and cell biology upon visualizing differential gene expression and protein localization and estimating protein association.

A mutation in ribosomal protein L9 markedly increases ribosomal hopping during translation of gene 60 from bacteriophage T4. This mutation, hop-1, converts Ser93 to Phe in rplI gene that encodes ribosomal large-subunit protein L9. The function of an RNA hairpin structure, which is crucial for ribosomal hopping, is replaced by this mutation.

Site-directed mutagenetic studies of the transmembrane segment S6 of domain IV (IVS6) in the sodium ion-channel reveals that the intracellular end of this segment contributes to fast channel inactivation. This segment is present in the hydrophobic receptor site for the fast inactivation gate. The entry to and stability of the inactivated state are maintained by three hydrophobic amino acid residues at sites 1774-1776 in the IVS6 segment.