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PubMed1995

Nucleotide sequence and chromosomal localization of the gene encoding the Old Yellow Enzyme from Kluyveromyces lactis.

A 6.6 kb genomic DNA fragment from the yeast Kluyveromyces lactis was isolated. Sequence analysis of this fragment revealed the presence of two incomplete open reading frames (ORFs) in one strand, one coding for the carboxyl terminus of the plasma membrane H(+)-ATPase and the other for the amino terminus of an unidentified product. In the complementary strand, a full-length ORF which encodes for a protein homologous to the yeast NADPH-dependent Old Yellow Enzyme was found. The deduced amino acid sequence of this ORF predicts a protein of 398 residues with 84% similarity in its full length to OYE1 from Saccharomyces carlsbergensis and OYE2 from Saccharomyces cerevisiae. In addition, an internal region showed considerable similarity to the bile acid-inducible polypeptide from Eubacterium sp., to the NADH oxidase from Thermoanaerobium brockii, to the trimethylamino dehydrogenase from bacterium W3A1 and to the estrogen-binding protein from Candida albicans, suggesting a functional or structural relationship between them. Inactivation of the KYE1 (Kluyveromyces Yellow Enzyme) gene by deletion of 0.6 kb fragment between positions +358 and +936 produced viable cells with a slight increase in their generation time. Haploid cells carrying the disrupted allele showed one-third of the NADPH oxidase activity, compared to wild-type cells. Southern blotting analysis of digested DNA and chromosomes separated by contour-clamped homogeneous electric field electrophoresis from K. lactis indicated that this is a single-copy gene and it is localized on chromosome II, whose molecular size has been estimated to be approximately 1.3 Mb.

Amino Acid Sequence↗
PubMed1987

Isolation of anonymous DNA sequences from within a submicroscopic X chromosomal deletion in a patient with choroideremia, deafness, and mental retardation.

Choroideremia, an X-chromosome linked retinal dystrophy of unknown pathogenesis, causes progressive nightblindness and eventual central blindness in affected males by the third to fourth decade of life. Choroideremia has been mapped to Xq13-21 by tight linkage to restriction fragment length polymorphism loci. We have recently identified two families in which choroideremia is inherited with mental retardation and deafness. In family XL-62, an interstitial deletion in Xq21 is visible by cytogenetic analysis and two linked anonymous DNA markers, DXYS1 and DXS72, are deleted. In the second family, XL-45, an interstitial deletion was suspected on phenotypic grounds but could not be confirmed by high-resolution cytogenetic analysis. We used phenol-enhanced reassociation of 48,XXXX DNA in competition with excess XL-45 DNA to generate a library of cloned DNA enriched for sequences that might be deleted in XL-45. Two of the first 83 sequences characterized from the library were found to be deleted in probands from family XL-45 as well as from family XL-62. Isolation of these sequences proves that XL-45 does contain a submicroscopic deletion and provides a starting point for identifying overlapping genomic sequences that span the XL-45 deletion. Each overlapping sequence will be studied to identify exons from the choroideremia locus.

Chromosome Mapping↗
PubMed1996

Compartmentalization of genes coding for immunodominant antigens to fragile chromosome ends leads to dispersed subtelomeric gene families and rapid gene evolution in Plasmodium falciparum.

Recent studies on the chromosome structure of Plasmodium falciparum have led to two observations: chromosome breakage occurs frequently in subtelomeric regions and the genes coding for a number of immunodominant parasite proteins are located in these fragile chromosomal segments. Toward understanding the biological significance of these observations, we have been studying the variability of a number of these telomeric genes in parasite lines isolated in different regions of the world. In this report, we present evidence that the telomeric location of the resa and the gbp genes of P. falciparum has allowed their dispersion to other chromosomes and eventual alteration. In the first example it is shown that the resa gene has been dispersed to subtelomeric positions on chromosomes 1, 2, 11 and 14 in clinical isolates from West African patients, giving rise to new parasite genotypes and gene linkage groups. Cloning and molecular analysis of the newly detected resa-related sequences reveal that two of the members of the family have diverged from the ancestral copy on chromosome 1, while the third member on chromosome 14 is very homologous to the ancestral copy indicating that it arose from a recent translocation event. In the second example, we show that the gbp genes form a dispersed gene family that maps to at least three different chromosome extremities. The data suggest that the compartmentalization of P. falciparum antigen genes to the chromosome ends lead to gene families scattered on several chromosome extremities. We propose that the generation of segmental aneuploidy is a specific mechanism of genome adaptation of P. falciparum to its host environment. We present a model to explain the duplicative translocation of chromosome termini.

Amino Acid Sequence↗
PubMed1992

Mutually exclusive exon splicing of the cardiac calcium channel alpha 1 subunit gene generates developmentally regulated isoforms in the rat heart.

Several clones were isolated from a rat genomic library in order to further characterize a region of variability within the third membrane-spanning region of the fourth motif (IVS3) of the L-type voltage-dependent calcium channel. We report here that this diversity arises from alternative splicing of a primary transcript containing a single pair of adjacent exons each encoding a unique sequence for the IVS3 region. Definitive proof of a mutually exclusive splicing mechanism was obtained by genomic mapping of flanking upstream and downstream exons and by extensive sequence analysis of the relevant exon/intron boundaries. S1 nuclease protection experiments revealed that both variant forms of the IVS3 were equally expressed in newborn and fetal rat heart, whereas only a single isoform predominated in adult rat heart. The results demonstrate the existence of an important developmentally regulated switch mediated by alternatively spliced exons in cardiac tissue at a time when major changes in excitation occur.

Amino Acid Sequence↗
PubMed2000

Genomic structure and chromosome location of the gene encoding mouse CD59.

The gene encoding the mouse analogue of the human complement regulator CD59 was cloned using a combination of long range PCR and genomic library screening. Sequence obtained showed that its genomic structure closely resembled that of the human CD59 gene, comprising 4 exons, each separated by a long intron region. The sizes of introns and exons were comparable to those of the human gene with the exception of the third intron which is 2.5 kb in the mouse compared to 7 kb in the human gene. All exon/intron boundaries conformed to the GT-AG rules for splicing. Radiation hybrid mapping localised mouse Cd59 between D2Mit333 and D2Mit127 on chromosome 2, a region homologous with human chromosome 11p13 where the human CD59 gene is localised. These data have permitted the construction of a gene targeting vector for the generation of transgenic mice deficient in CD59.

Animals↗
PubMed2005

Green sperm. Identification of male gamete promoters in Arabidopsis.

Previously, in an effort to better understand the male contribution to fertilization, we completed a maize (Zea mays) sperm expressed sequence tag project. Here, we used this resource to identify promoters that would direct gene expression in sperm cells. We used reverse transcription-polymerase chain reaction to identify probable sperm-specific transcripts in maize and then identified their best sequence matches in the Arabidopsis (Arabidopsis thaliana) genome. We tested five different Arabidopsis promoters for cell specificity, using an enhanced green fluorescent protein reporter gene. In pollen, the AtGEX1 (At5g55490) promoter is active in the sperm cells and not in the progenitor generative cell or in the vegetative cell, but it is also active in ovules, roots, and guard cells. The AtGEX2 (At5g49150) promoter is active only in the sperm cells and in the progenitor generative cell, but not in the vegetative cell or in other tissues. A third promoter, AtVEX1 (At5g62850) [corrected] was active in the vegetative cell during the later stages of pollen development; the other promoters tested (At1g66770 and At1g73350) did not function in pollen. Comparisons among GEX1 and GEX2 homologs from maize, rice (Oryza sativa), Arabidopsis, and poplar (Populus trichocarpa) revealed a core binding site for Dof transcription factors. The AtGEX1 and AtGEX2 promoters will be useful for manipulating gene expression in sperm cells, for localization and functional analyses of sperm proteins, and for imaging of sperm dynamics as they are transported in the pollen tube to the embryo sac.

Arabidopsis↗
PubMed1991

Structure of the mouse pore-forming protein (perforin) gene: analysis of transcription initiation site, 5' flanking sequence, and alternative splicing of 5' untranslated regions.

We studied the 5' untranslated regions (UTRs) of the mouse lymphocyte pore-forming protein (PFP, perforin, and cytolysin). 5' UTRs were determined by primer extension analysis, sequencing PFP cDNA clone PFP-7, ribonuclease protection assays, and amplification of poly(A)+ RNA of cytolytic T lymphocyte using polymerase chain reaction (PCR). Two alternatively spliced 5' UTRs, designated type I and type II, of 222 and 115 bp, respectively, were found associated with PFP. Type II is identical to type I, except for being 107 bp shorter in the second exon. This deletion was generated by the use of alternative acceptor splice sites. The mouse PFP gene (Pfp) encodes three exons, is separated by two small introns, and spans a chromosomal region of approximately 7 kb. The first exon contains 79 bp of 5' UTR, the second exon contains 143 or 36 bp of 5' UTR (type I or type II UTR, respectively) plus the NH2-terminal region of the mouse PFP, and the third exon contains the rest of the COOH-terminal mouse PFP. The organization of the mouse Pfp is similar to that of the human gene. Moreover, the 5' flanking sequence of the mouse Pfp is highly homologous to that of the human Pfp. In contrast to the human sequence, the more immediate 5' flanking sequence of mouse Pfp contains two tandem "TATA" box-related elements and a GC box, but lacks a typical CAAT box-related sequence. Several other enhancer elements were found further upstream, including cAMP-, phorbol ester-, interferon-gamma-, and UV-responsive elements, and PU box-like and NFkB binding site-like elements. In addition, we found a nuclear inhibitory protein-like element, a transcriptional silencer, and a pair of purine-rich sequence motifs that were found in other T cell-specific genes, and three repeats of GGCCTG that may be a variation of a highly repetitious GCCCTG consensus sequence found in human Pfp.

Animals↗
PubMed1988

Maturation of rat visual cortex: IV. The generation, migration, morphogenesis, and connectivity of atypically oriented pyramidal neurons.

The generation, migration, and morphogenesis of atypically oriented pyramidal neurons in the rat visual cortex were examined. In the mature cortex, these neurons were distributed through layers II-VI. Moreover, the atypically oriented pyramidal neurons in a particular layer tended to be oriented in a specific way; atypically oriented pyramidal neurons in layer II, layers III-VIa, and layer VIb were obliquely, radially, and obliquely oriented, respectively. Ultrastructurally, the somata of atypically oriented pyramidal neurons contained large euchromatic ovoid nuclei and cytoplasm that was replete with rough endoplasmic reticulum and Golgi apparatus. These somata formed only symmetric axosomatic synapses. Many atypically oriented pyramidal neurons projected axons into the white matter as demonstrated by a Golgi method and by a retrograde tract-tracing technique; however, some of these pyramidal neurons in layers III-V had axons that ascended to layer I. By using a technique which combined retrograde tract tracing with [3H]thymidine autoradiography, it was determined that most atypically oriented pyramidal neurons in layers V and VIa, layer IV, and layer II were generated on gestational days (GD) 15-17, GD 17-19, and GD 20-21, respectively. Atypically oreinted pyramidal neurons were identified during the period from postnatal day 0 (day of birth) to day 30. On day 0, obliquely oriented pyramidal neurons were distributed in the deep cortical plate, i.e., immature layer VI. On day 3, the youngest atypically oriented pyramidal neurons were radially oriented and were located in layer IV. Some obliquely oriented pyramidal neurons were present in layer II on day 6, but the greatest number and the most severely canted pyramidal neurons in layer II were evident on day 9. The orientations of the cell body and the apical dendrite did not change appreciably after migration was complete, except for those in layers V and VI with obliquely oriented cell bodies and radially oriented apical dendrites. The second and third postnatal weeks were marked by substantial morphological differentiation of all pyramidal neurons as noted by the lengthening and branching of dendrites and by the appearance of dendritic spines. By the fourth postnatal week, atypically oriented pyramidal neurons achieved their mature morphology. The generation, migration, and morphogenesis of atypically oriented pyramidal neurons proceed by an inside-to-outside sequence. This development is similar and concurrent with that of typically oriented pyramidal neurons.

Aging↗
PubMed2006

A mutation c.C2812T in the androgen receptor gene resulting in Pro817Leu substitution may affect dimerization of the androgen receptor and result in androgen insensitivity syndrome.

OBJECTIVE: To conduct clinical, genetic, and molecular diagnostics of two sisters with typical symptoms of complete androgen insensitivity syndrome. DESIGN: Case report. SETTING: Research laboratory at a university of medical science. PATIENT(S): Two patients with 46,XY karyotype and a female phenotype were diagnosed because of primary amenorrhea. Their sister with 46,XX karyotype, her daughter, and five other family members including their mother also were examined. INTERVENTION(S): Orchiectomy, estrogen substitution therapy. MAIN OUTCOME MEASURE(S): Cancer prophylaxis. RESULT(S): Multiple-temperature single-stranded conformation polymorphism and sequence analyses of the androgen receptor gene (AR) revealed a c.C2812T transition in exon 7 in the two sisters. Their mother and the third sister (46,XX) were carriers of the same mutation. This mutation, which previously had never been reported, resulted in Pro817Leu substitution in the ligand-binding domain of the androgen. Computer simulation of structural changes generated by Pro817Leu substitution revealed appreciable conformational changes in the region responsible for dimerization of the receptor. CONCLUSION(S): The novel c.C2812T transition that might impair dimerization of the receptor is responsible for the clinical symptoms of complete androgen insensitivity syndrome in the affected individuals. Molecular analysis of AR proved to be very useful for genetic counseling of the unaffected sister, who was a carrier of the same mutation.

Adult↗
PubMed1997

A unique molecular basis for enzyme mistargeting in primary hyperoxaluria type 1.

The intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is normally targeted to the peroxisomes in human liver cells. However, in a third of patients suffering from the autosomal recessive disease primary hyperoxaluria type 1 (PH1), AGT is mistargeted to the mitochondria. Such organelle-to-organelle mistargeting is without parallel in human genetic disease. AGT mistargeting results from the combination of a common Pro11-->Leu polymorphism and a rare Gly170-->Arg mutation. The former generates a functionally weak mitochondrial targeting sequence (MTS) while the latter, in combination with the former, increases the efficiency of this MTS by slowing the rate at which AGT dimerises. The fact that the intracellular compartmentation of AGT can be determined, at least in part, by its oligomeric status highlights the fundamental differences in the molecular requirements for protein import into two intracellular organelles--the peroxisomes and mitochondria.

Amino Acids↗
PubMed2000

Seed plant phylogeny inferred from all three plant genomes: monophyly of extant gymnosperms and origin of Gnetales from conifers.

Phylogenetic relationships among the five groups of extant seed plants are presently quite unclear. For example, morphological studies consistently identify the Gnetales as the extant sister group to angiosperms (the so-called "anthophyte" hypothesis), whereas a number of molecular studies recover gymnosperm monophyly, and few agree with the morphology-based placement of Gnetales. To better resolve these and other unsettled issues, we have generated a new molecular data set of mitochondrial small subunit rRNA sequences, and have analyzed these data together with comparable data sets for the nuclear small subunit rRNA gene and the chloroplast rbcL gene. All nuclear analyses strongly ally Gnetales with a monophyletic conifers, whereas all mitochondrial analyses and those chloroplast analyses that take into account saturation of third-codon position transitions actually place Gnetales within conifers, as the sister group to the Pinaceae. Combined analyses of all three genes strongly support this latter relationship, which to our knowledge has never been suggested before. The combined analyses also strongly support monophyly of extant gymnosperms, with cycads identified as the basal-most group of gymnosperms, Ginkgo as the next basal, and all conifers except for Pinaceae as sister to the Gnetales + Pinaceae clade. According to these findings, the Gnetales may be viewed as extremely divergent conifers, and the many morphological similarities between angiosperms and Gnetales (e.g., double fertilization and flower-like reproductive structures) arose independently.

Cycadopsida↗
PubMed2006

Chromosomal translocations fusing the BCL6 gene to different partner loci are recurrent in primary central nervous system lymphoma and may be associated with aberrant somatic hypermutation or defective class switch recombination.

Primary central nervous system lymphomas (PCNSLs) are diffuse large B cell lymphomas confined to the brain. Only minimal data exist on chromosomal aberrations underlying PCNSLs. We studied 41 PCNSLs by fluorescence in situ hybridization for breakpoints affecting the BCL6 locus in chromosomal band 3q27. Of 37 cases evaluable, 14 (38%) carried a breakpoint in the BCL6 locus. Two of these showed juxtaposition of BCL6 to the IGH locus. In 4 cases, the BCL6 breakpoints were cloned using long-distance inverse polymerase chain reaction. All breakpoints were located within the BCL6 major translocation cluster. The translocation partners were the IGH gene in 14q32.33, the IGL gene in 22q11.22, and the histone 1 H4I gene in 6p22.1. In the fourth case, a deletion in 3q leads to loss of an 837-kb fragment extending from the first intron of BCL6 to the third intron of the lipoma-preferred partner (LPP) gene. This deletion may bring the BCL6 gene under the control of regulatory elements of the LPP gene or the miRNA-28 gene located in intron 4 of LPP. DNA sequence analysis of the junctional sequences provided evidence that aberrant class switch recombination or somatic hypermutation may be involved in the generation of BCL6 translocations.

Base Sequence↗
PubMed1987

Amino acid sequence of UP1, an hnRNP-derived single-stranded nucleic acid binding protein from calf thymus.

The UP1 single-stranded nucleic acid binding protein from calf thymus (Herrick, G. & Alberts, B.M. (1976) J. Biol. Chem. 251, 2124-2132) has recently been shown to be a proteolytic fragment derived from the A1 heterogeneous nuclear ribonucleoprotein (hnRNP) (Pandolfo et al. (1985) Nucleic Acids Res. 13, 6577-6590). The NH2-terminus of the 22,162 dalton UP1 protein appears to be blocked, which suggests that UP1 represents the NH2-terminal two thirds of this 32,000 dalton hnRNP protein. The complete amino acid sequence for UP1 was derived from automated sequencing of peptides that were purified by HPLC from digests with trypsin, chymotrypsin, Staphylococcus aureus protease, endoproteinase Lys-C, and cyanogen bromide. Trichloroacetic acid precipitation followed by enzymatic digestion in 2 M urea proved to be the best approach for generating UP1 peptides. By carboxymethylating after, rather than before, digestion it was possible to avoid problems associated with the insolubility of the carboxymethylated UP1. All of the resulting peptides in amounts varying from 2 to 15 nmol were coupled to aminopolystyrene prior to solid-phase sequencing. Using these methods, no difficulties were encountered in assigning glutamic acid residues or in completely sequencing peptides that contained up to 25-30 residues. The relative ease with which the UP1 protein was sequenced, requiring only about a year to complete, and the comparatively modest amount of protein required, less than 5 mg, attests to the usefulness of water soluble carbodiimide coupling and solid-phase sequencing for determining the primary structures of proteins. In addition to serving as a basis for determining structural relationships among various mammalian single-stranded nucleic acid binding proteins, the amino acid sequence of UP1 reveals that the A1 hnRNP protein contains a region of internal sequence homology that apparently corresponds to two independent nucleic acid binding sites.

Amino Acid Sequence↗
PubMed1999

Conserved cytoplasmic loops are important for both the transport and chemotaxis functions of PcaK, a protein from Pseudomonas putida with 12 membrane-spanning regions.

Chemotaxis to the aromatic acid 4-hydroxybenzoate (4-HBA) by Pseudomonas putida is mediated by PcaK, a membrane-bound protein that also functions as a 4-HBA transporter. PcaK belongs to the major facilitator superfamily (MFS) of transport proteins, none of which have so far been implicated in chemotaxis. Work with two well-studied MFS transporters, LacY (the lactose permease) and TetA (a tetracycline efflux protein), has revealed two stretches of amino acids located between the second and third (2-3 loop) and the eighth and ninth (8-9 loop) transmembrane regions that are required for substrate transport. These sequences are conserved among most MFS transporters, including PcaK. To determine if PcaK has functional requirements similar to those of other MFS transport proteins and to analyze the relationship between the transport and chemotaxis functions of PcaK, we generated strains with mutations in amino acid residues located in the 2-3 and 8-9 loops of PcaK. The mutant proteins were analyzed in 4-HBA transport and chemotaxis assays. Cells expressing mutant PcaK proteins had a range of phenotypes. Some transported at wild-type levels, while others were partially or completely defective in 4-HBA transport. An aspartate residue in the 8-9 loop that has no counterpart in LacY and TetA, but is conserved among members of the aromatic acid/H(+) symporter family of the MFS, was found to be critical for 4-HBA transport. These results indicate that conserved amino acids in the 2-3 and 8-9 loops of PcaK are required for 4-HBA transport. Amino acid changes that decreased 4-HBA transport also caused a decrease in 4-HBA chemotaxis, but the effect on chemotaxis was sometimes slightly more severe. The requirement of PcaK for both 4-HBA transport and chemotaxis demonstrates that P. putida has a chemoreceptor that differs from the classical chemoreceptors described for Escherichia coli and Salmonella typhimurium.

Amino Acid Sequence↗
PubMed1998

A third-generation lentivirus vector with a conditional packaging system.

Vectors derived from human immunodeficiency virus (HIV) are highly efficient vehicles for in vivo gene delivery. However, their biosafety is of major concern. Here we exploit the complexity of the HIV genome to provide lentivirus vectors with novel biosafety features. In addition to the structural genes, HIV contains two regulatory genes, tat and rev, that are essential for HIV replication, and four accessory genes that encode critical virulence factors. We previously reported that the HIV type 1 accessory open reading frames are dispensable for efficient gene transduction by a lentivirus vector. We now demonstrate that the requirement for the tat gene can be offset by placing constitutive promoters upstream of the vector transcript. Vectors generated from constructs containing such a chimeric long terminal repeat (LTR) transduced neurons in vivo at very high efficiency, whether or not they were produced in the presence of Tat. When the rev gene was also deleted from the packaging construct, expression of gag and pol was strictly dependent on Rev complementation in trans. By the combined use of a separate nonoverlapping Rev expression plasmid and a 5' LTR chimeric transfer construct, we achieved optimal yields of vector of high transducing efficiency (up to 10(7) transducing units [TU]/ml and 10(4) TU/ng of p24). This third-generation lentivirus vector uses only a fractional set of HIV genes: gag, pol, and rev. Moreover, the HIV-derived constructs, and any recombinant between them, are contingent on upstream elements and trans complementation for expression and thus are nonfunctional outside of the vector producer cells. This split-genome, conditional packaging system is based on existing viral sequences and acts as a built-in device against the generation of productive recombinants. While the actual biosafety of the vector will ultimately be proven in vivo, the improved design presented here should facilitate testing of lentivirus vectors.

Animals↗
PubMed2006

Signal regulatory protein molecules are differentially expressed by CD8- dendritic cells.

A normalized subtracted gene expression library was generated from freshly isolated mouse dendritic cells (DC) of all subtypes, then used to construct cDNA microarrays. The gene expression profiles of the three splenic conventional DC (cDC) subsets were compared by microarray hybridization and two genes encoding signal regulatory protein beta (Sirpbeta1 and Sirpbeta4) molecules were identified as differentially expressed in CD8(-) cDC. Genomic sequence analysis revealed a third Sirpbeta member localized in the same gene cluster. These Sirpbeta genes encode cell surface molecules containing extracellular Ig domains and short intracytoplasmic domains that have a charged amino acid in the transmembrane region which can potentially interact with ITAM-bearing molecules to mediate signaling. Indeed, we demonstrated interactions between Sirpbeta1 and beta2 with the ITAM-bearing signaling molecule Dap12. Real-time PCR analysis showed that all three Sirpbeta genes were expressed by CD8(-) cDC, but not by CD8(+) cDC or plasmacytoid pre-DC. The related Sirpalpha gene showed a similar expression profile on cDC subtypes but was also expressed by plasmacytoid pre-DC. The differential expression of Sirpalpha and Sirpbeta1 molecules on DC was confirmed by staining with mAbs, including a new mAb recognizing Sirpbeta1. Cross-linking of Sirpbeta1 on DC resulted in a reduction in phagocytosis of Leishmania major parasites, but did not affect phagocytosis of latex beads, perhaps indicating that the regulation of phagocytosis by Sirpbeta1 is a ligand-dependent interaction. Thus, we postulate that the differential expression of these molecules may confer the ability to regulate the phagocytosis of particular ligands to CD8(-) cDC.

Amino Acid Sequence↗
PubMed1991

Rat liver polysome N alpha-acetyltransferase: substrate specificity.

The substrate specificity of polysome rat liver N alpha-acetyltransferase (NAT) has been examined by utilizing a series of synthetic and natural substrates that has been systematically altered with respect to N-terminal sequence and length. Families of peptides of the structure S-Y-S-G-G-L-L-L were generated by successively replacing the N-terminal serine, the penultimate tyrosine, and the antepenultimate serine with all 19 commonly occurring amino acids, which were then assessed for their reactivity with the rat liver enzyme. Only peptides with N-terminal serine, alanine, methionine, leucine, and phenylalanine were modified. Glycine, lysine, arginine, valine, isoleucine, and tryptophan in the second position are (with N-terminal serine) strongly inhibitory, and proline completely blocks modification. Third-position substitutions have less of an effect on NAT activity with glycine, aspartic acid, glutamic acid, and tryptophan being most inhibiting (with N-terminal Ser-Tyr). These observations are generally in agreement with in situ modifications although there are some significant differences particularly with respect to the amino-terminal residues. Optimal chain length was determined to be 10-11 residues with either synthetic peptides of the structure S-Y-S-(G)n-L-L-L or adrenocorticotropin (ACTH) sequences ranging from 8 to 39 residues. The ACTH peptides were generally found to be severalfold better substrates than the corresponding synthetic ones. Activity was not affected by increased chain length beyond approximately 17 residues. These data support the view that polysome-catalyzed N alpha-acetylation occurs as a cotranslational event on nascent chains of about 20-40 amino acids in length.

Acetylation↗
PubMed1997

Identification of mutations in two major mRNA isoforms of the Chediak-Higashi syndrome gene in human and mouse.

Chediak-Higashi syndrome is an autosomal recessive, immune deficiency disorder of human (CHS) and mouse (beige, bg) that is characterized by abnormal intracellular protein transport to, and from, the lysosome. Recent reports have described the identification of homologous genes that are mutated in human CHS and bg mice. Here we report the sequences of two major mRNA isoforms of the CHS gene in human and mouse. These isoforms differ both in size and in sequence at the 3' end of their coding domains, with the smaller isoform (approximately 5.8 kb) arising from incomplete splicing and reading through an intron. These mRNAs also differ in tissue distribution of transcription and in predicted biological properties. Novel mutations were identified within the region of the coding domain common to both isoforms in three CHS patients: C-->T transitions that generated stop codons (R50X and Q1029X) were found in two patients, and a novel frameshift mutation (deletion of nucleotides 3073 and 3074 of the coding domain) was found in a third. Northern blots of lymphoblastoid mRNA from CHS patients revealed loss of the largest transcript (approximately 13.5 kb) in two of seven CHS patients, while the small mRNA was undiminished in abundance. These results suggest that the small isoform alone cannot complement Chediak-Higashi syndrome.

Alternative Splicing↗