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PubMed1991

Mapping using unique sequences.

Theoretical predictions are given for the progress expected, when mapping DNA by identifying clones containing specific unique sequences. Progress is measured in three ways; however, all results depend on (dimensionless counterparts of) the number of clones and the number of unique sequences used. Furthermore, the effects of clone length dispersion are included in the theoretical predictions. Both the clones in the library and the unique sequences are assumed to be generated randomly, with uniform probability of originating at any base in the region to be mapped. The first measure of progress is the expected length fraction of the region to be mapped covered by at least one clone, when clones containing at least one unique sequence are included in the map. The second measure of progress is the expected length fraction of the region to be mapped in "covered intervals", an interval being the region between adjacent unique sequences. Alternative definitions for clones covering an interval are analyzed. The third measure of progress is the expected number of clone islands generated; an island covers successive intervals. Finally, using these measures of progress, we compare the efficiency of this new mapping strategy with conventional clone mapping strategies.

Chromosome Mapping↗
PubMed1997

Porcine aromatases: studies on tissue-specific, functionally distinct isozymes from a single gene?

Aromatase cytochrome P450 (P450arom) is expressed in a variety of tissues. Pigs express P450arom as bilaminar blastocysts in utero, and thereafter in the gonads, adrenal glands and placenta. Our studies also demonstrate the existence of porcine isozymes of P450arom which differ substantially in their amino acid composition and function. The placental isoform, most similar to P450arom in other mammals, consists of 503 amino acids. The ovarian isoform, expressed in both theca and granulosa cells, is a 501 amino acid protein exhibiting less than 20% of the activity of the placental isozyme. Furthermore, it is inhibited not only by CGS16949A but also by etomidate which does not inhibit the placental P450arom. Partial sequences generated by the rapid amplification of the cDNA ends (RACE) procedure indicate that the expression of a third isoform in the blastocyst is switched to the placental isozyme during differentiation of the fetal membranes. In addition, these transcripts, and others from the theca, granulosa, testes, adrenal glands and placenta demonstrate differences in the 5'-untranslated region (putative exon I) suggestive of tissue-specific alternative splicing. An identical 5'-untranslated sequence was obtained from transcripts expressed in the theca and granulosa. Testes and adrenal transcripts also have identical 5' ends, which differ substantially from the ovarian sequence. Blastocyst and placenta 5'-untranslated sequences differ from each other and from those expressed in the gonads and adrenals. Several tissue-specific transcripts thus encode porcine P450arom. Interestingly, distinct 5' sequences exist for ovarian and testes P450arom mRNAs, suggesting different promoters and therefore regulation in the male and female gonads. The molecular origins of the functional isoforms and the tissue-specific transcripts are uncertain, however partial genomic sequence and other genetic analyses suggest the existence of multiple genes. However, sequence alignment of the placental and ovarian isoforms indicates complete conservation of putative exon III, so that complex splicing remains a possibility. Clearly, the regulation of P450arom expression is more complex in the pig than in other vertebrates investigated to date.

Amino Acid Sequence↗
PubMed1995

Structural organization of the murine D3 dopamine receptor gene.

We have cloned the gene encoding the murine D3 dopamine receptor and have analyzed its intron-exon structural organization, to gain a better understanding of the detailed architecture of the D2 dopamine receptor genes. Restriction and sequence analysis reveal the presence of six introns, in contrast to the five introns previously reported for the rat D3 receptor. The extra intron is located in the receptor's putative third cytoplasmic loop and generates an intron-exon organization directly analogous to that found in the D2 receptor gene. In addition, we have sequenced the 5' and 3' nontranslated sequences flanking the coding region and have identified a putative poly(A) adenylation signal. These sequences are found to have a far lower homology with the corresponding rat nontranslated sequences than is found for the D2 receptor, suggesting that the control of D3 receptor expression may vary more between species than the control of D2 receptor expression.

Amino Acid Sequence↗
PubMed2000

Emergence of imipenem resistance in Klebsiella pneumoniae owing to combination of plasmid-mediated CMY-4 and permeability alteration.

Klebsiella pneumoniae BM2974 isolated from an abdominal abcess was resistant to high concentrations of all available beta-lactams, including recently developed third-generation cephalosporins and carbapenems. Isoelectric focusing of beta-lactamases and amplification, cloning and sequencing of the corresponding genes, together with conjugation and transformation experiments, indicated that, in addition to the chromosomally encoded beta-lactamase, the strain produced three plasmid-mediated beta-lactamases with pIs of 5.4, 8.2 and 9.0, which corresponded to TEM-1, SHV-5 and AmpC-type CMY-4, respectively. Strain BM2974 also lacked a major outer membrane protein of c. 40 kDa which was present in the spontaneous imipenem-susceptible revertant BM2974-1. We suggest that imipenem resistance in strain BM2974 is attributable to production of CMY-4 beta-lactamase combined with permeability alteration.

Bacterial Outer Membrane Proteins↗
PubMed1999

Autoregressive moving average modeling for finger tapping with an external stimulus.

We report the Autoregressive Moving Average (ARMA) structures of interresponse interval and interstimulus interval in rhythmic finger tapping and how tapping schemes arise from these structures. The interstimulus interval is chosen to be a metronome cue slightly perturbed from its periodic mode by a zero-mean sine-wave modulated sequence. These metronome sequences were chosen to follow an exact Autoregressive model of the third order and are generated using metronome interval durations of 400, 500, and 600 msec. At each frequency, four different perturbation rates were presented to seven subjects: 1, 3, 5, and 7% of the base interval. Synchronization error, i.e., tap-to-beat phase deviations, induced by these ARMA models are stable and thus were bounded. This framework is useful in characterizing the relationship between motor control variables, interresponse interval, synchronization error, and interstimulus interval. Also, several known models can be easily derived and analyzed in this setting.

Adult↗
PubMed1995

An engineered Staphylococcus aureus PC1 beta-lactamase that hydrolyses third-generation cephalosporins.

The beta-lactamase from Staphylococcus aureus PC1 has been cloned into an Escherichia coli vector for site-directed mutagenesis and high-level protein expression. A mutant enzyme has been produced in which Ala238 is replaced by a serine, and Ile239 is deleted (A238S:I239del). The engineered enzyme hydrolyses third-generation cephalosporins substantially more rapidly than the parental enzyme does, while hydrolysis of benzylpenicillin is slower with the mutant than with the wild-type and native enzymes. The mutant beta-lactamase has been crystallized and the structure determined and refined at 2.8 A resolution. The disposition of the beta-strand which forms the side of the active site is altered in comparison with the native S. aureus beta-lactamase structure, widening the active site cleft and providing space to accommodate the bulky side-chains of the third-generation cephalosporins.

Base Sequence↗
PubMed1986

Mutational analysis of bacteriophage lambda lysis gene S.

A plasmid carrying the bacteriophage lambda lysis genes under lac control was subjected to hydroxylamine mutagenesis, and mutations eliminating the host lethality of the S gene were selected. DNA sequence analysis revealed 48 single-base mutations which resulted in alterations within the coding sequence of the S gene. Thirty-three different missense alleles were generated. Most of the missense changes clustered in the first two-thirds of the molecule from the N terminus. A simple model for the disposition of the S protein within the inner membrane can be derived from inspection of the primary sequence. In the first 60 residues, there are two distinct stretches of predominantly hydrophobic amino acids, each region having a net neutral charge and extending for at least 20 residues. These regions resemble canonical membrane-spanning domains. In the model, the two domains span the bilayer as a pair of net neutral charge helices, and the N-terminal 10 to 12 residues extend into the periplasm. The mutational pattern is largely consistent with the model. Charge changes within the putative imbedded regions render the protein nonfunctional. Loss of glycine residues at crucial reverse-turn domains which would be required to reorient the molecule to reenter the membrane also inactivate the molecule. Finally, a number of neutral and rather subtle mutations such as Ala to Val and Met to Ile are found, mostly within the putative spanning regions. Although no obvious explanation exists for this subtle and heterogeneous class of mutations, it is noted that all of the changes result in a loss of alpha-helical character as predicted by Chou-Fasman theoretical analysis. Alternative explanations for some of these changes are also possible, including a reduction in net translation rate due to substitution of a rare codon for a common one. The model and the pattern of mutations have implications for the probable oligomerization of the S protein at the time of endolysin release at the end of the vegetative growth period.

Alleles↗
PubMed2001

cAMP-dependent phosphorylation of CYP2B1 as a functional switch for cyclophosphamide activation and its hormonal control in vitro and in vivo.

An important feature of cytochrome P450 (CYP) 2B1 is its high ability to convert the prodrug cyclophosphamide (CPA) to therapeutically cytotoxic metabolites, resulting in interstrand DNA-cross-linking and cell death. We have examined whether and how the phosphorylation of CYP2B1 influences CPA metabolic activation in vitro and in vivo. We found first that only part of the total CYP2B1 pool undergoes phosphorylation. This part is fully inactivated. Second, phosphorylation of CYP2B1 in intact hepatocytes reduced by up to 75% toxification of CPA to mutagenic metabolites (totally dependent on the same preferentially CYP2B-catalyzed 4-hydroxylation of CPA as is the generation of highly cytotoxic species). Third, the phosphoacceptor-serine 128 of CYP2B1 in the consensus sequence for interaction with the protein kinase A represents an on/off switch for the activation of CPA depending on the phosphorylation conditions in the cell. Fourth, evidence is presented that the above-described events also occur in vivo. In conclusion, a successful therapy with CPA, helped by forced expression of CYP2B1 in tumor cells (as recently proposed) will, in addition, be profoundly modified by its phosphorylation status.

Animals↗
PubMed1988

Lessons drawn from observing young peers together.

Based on studies during the past 13 years of what transpires between young peers, lessons are drawn about the nature of human sociability and the development of social skill during the first three years of life. Peer encounters have proven especially helpful for discovering the forms of sociability and social skill the infant is capable of without the aid of a more skillful social partner. From early in infancy, children are quite sociable with peers (age-mates), both in novel play settings and in their own home or customary group care settings, both with an unfamiliar peer and with those quite familiar, both at the start of acquaintanceship with a particular peer and after many encounters with that peer. Their sociability is seen in their attraction to peers, their directing to peers of such distinctively social behaviors as vocalizations, smiles, and gestures, and the predominantly friendly nature of their behavior. Peer encounters in the absence of customary play materials refute claims that attraction to peers is a by-product of interest in toys and the inanimate spectacles peers create through their actions on toys. Distinctions should be drawn between sociability and social skills, especially interactive skills. Interactive skills are systematic ways the young child relates his/her own behavior to the details of a partner's behavior that function to facilitate such valued social outcomes as the generation of a cooperative game or a conversation or the resolution of a dispute. Observations of young peers highlight the distinctive nature of the infant's interactive skills. Social influence between peers is present from 6 months, but rarely takes the form customary for older children. Joining a peer and manipulating the same play material as the peer are rudimentary interactive skills that emerge by 12 months. Interactive skills enabling the generation of extended sequences of social interaction on a common cooperative theme progress rapidly during the third year of life. The distinctive form of the young child's interactive skills produces distinctive patterns of interaction among young peers. Their interactions are managed largely nonverbally well into the third year of life, and extended cooperative interactions most often take the form of games in which one or both children imitate each other's actions. Despite the predominance of imitation, their encounters are also marked by complementary role relationships. The distinctive features of peer encounters prompt speculations about the role peer encounters can play in early development.

Child, Preschool↗
PubMed1992

Cytotoxic T lymphocytes do not appear to select for mutations in an immunodominant epitope of simian immunodeficiency virus gag.

Studies to date assessing HIV escape from CTL in vivo have yielded conflicting results. Previous studies have demonstrated that simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys expressing the MHC class I allele Mamu-A*01 reproducibly develop a gag-specific CTL response limited to a 9-amino acid epitope of the SIVmac gag protein (residues 182-190 within peptide 11C). To determine whether CTL have a role in selecting for AIDS virus mutants, we examined mutations in SIVmac proviral DNA encoding this gag CTL epitope in PBL of infected rhesus monkeys. Three Mamu-A*01+ rhesus monkeys were infected with SIVmac and assessed for gag- and peptide 11C-specific CTL responses. This specific CTL response was maintained in two monkeys, but lost in the third animal 2 yr after infection. The generation of proviral gag mutations was then determined by sequencing 500-bp proviral fragments amplified from fresh PBL obtained from the monkeys more than 2.5 yr after infection. Although numerous point mutations were characterized in 131 polymerase chain reaction-generated clones of SIVmac gag, only four mutations within the gag CTL epitope-coding region of the genome were identified. Comparison of synonymous and nonsynonymous nucleotide substitutions in the regions encoding peptide 11C (p11C) and the flanking gag protein indicated a lack of selective pressure for viral mutations in the CTL epitope coding region. Interestingly, a predominant gag mutant encoding a single amino acid change in p11C was found in a monkey which lost its CTL activity. However, even in this setting there was no evidence for selection of mutations in the CTL epitope coding region when compared with the flanking region. Furthermore, synthetic peptides corresponding to all naturally occurring variants in the gag epitope-coding region were recognized by cloned and bulk cultured effector cells of the infected monkeys with persistent CTL. These results indicate that SIVmac gag- and p11C-specific CTL do not select for mutations in the immunodominant epitope-coding region and that the naturally occurring mutants do not appear to escape CTL recognition.

Amino Acid Sequence↗
PubMed2001

Words in a sea of sounds: the output of infant statistical learning.

One of the first problems confronting infant language learners is word segmentation: discovering the boundaries between words. Prior research suggests that 8-month-old infants can detect the statistical patterns that serve as a cue to word boundaries. However, the representational structure of the output of this learning process is unknown. This research assessed the extent to which statistical learning generates novel word-like units, rather than probabilistically-related strings of sounds. Eight-month-old infants were familiarized with a continuous stream of nonsense words with no acoustic cues to word boundaries. A post-familiarization test compared the infants' responses to words versus part-words (sequences spanning a word boundary) embedded either in simple English contexts familiar to the infants (e.g. "I like my tibudo"), or in matched nonsense frames (e.g. "zy fike ny tibudo"). Listening preferences were affected by the context (English versus nonsense) in which the items from the familiarization phase were embedded during testing. A second experiment confirmed that infants can discriminate the simple English contexts and the matched nonsense frames used in Experiment 1. The third experiment replicated the results of Experiment 1 by contrasting the English test frames with non-linguistic frames generated from tone sequences. The results support the hypothesis that statistical learning mechanisms generate word-like units with some status relative to the native language.

Auditory Perception↗
PubMed1997

Site-specific crosslinking of mammalian U11 and u6atac to the 5' splice site of an AT-AC intron.

A rare class of introns with AT-AC at their termini recently has been identified in metazoan genes. Splicing of these introns requires a different set of small nuclear ribonucleoprotein particles (snRNPs) (U11, U12, U5, and U4atac/U6atac) compared with the snRNPs (U1, U2, U5, and U4/U6) required for splicing the majority of pre-mRNA introns, but otherwise little is known regarding the excision of AT-AC introns. Here we use site-specific 4-thiouridine (4SU) crosslinking analysis to dissect the mechanism of 5' splice site recognition during in vitro splicing of the AT-AC intron from the P120 pre-mRNA. Upon irradiation with 365-nm UV light, three P120 substrates, each with a single 4SU substitution near the 5' splice site (at position +2, +4, or +7), produce two early ATP-independent crosslinks with similar kinetics. For one of the substrates, P120-4SU+2, a third ATP-requiring crosslink forms as the two early crosslinks diminish. RNase H digestion coupled with Northern blotting indicates that the two early crosslinks generated with P120-4SU+2 contain the U11 small nuclear RNA. Reverse transcription-PCR followed by cloning and sequencing demonstrates that the third crosslink involves U6atac. The dynamic appearance of the three crosslinks correlates with the kinetics of the splicing reaction and suggests that the 5' splice site is recognized first by U11 and then by U6atac. Our results argue that the splicing of AT-AC introns is mechanistically similar to the splicing of the major class of introns and that the U11 and U6atac snRNPs in the AT-AC spliceosome fulfill analogous roles to U1 and U6, respectively, in the major spliceosome.

Adenine↗
PubMed1989

Structural requirements for membrane assembly of proteins spanning the membrane several times.

We have investigated the structural requirements for the biogenesis of proteins spanning the membrane several times. Proteins containing various combinations of topological signals (signal anchor and stop transfer sequences) were synthesized in a cell-free translation system and their membrane topology was determined. Proteins spanning the membrane twice were obtained when a signal anchor sequence was followed by either a stop transfer sequence or a second signal anchor sequence. Thus, a signal anchor sequence in the second position can function as a stop transfer sequence, spanning the membrane in the opposite orientation to that of the first signal anchor sequence. A signal anchor sequence in the third position was able to insert amino acid sequences located COOH terminal to it. We conclude that proteins spanning the membrane several times can be generated by stringing together signal anchor and stop transfer sequences. However, not all proteins with three topological signals were found to span the membrane three times. A certain segment located between the first and second topological signal could prevent stable membrane integration of a third signal anchor segment.

Cell Membrane↗
PubMed1993

Cloning and structural determination of human peptide YY cDNA and gene.

We have isolated two kinds of cDNAs and the gene encoding human peptide YY and determined their nucleotide sequences. The human peptide YY gene is composed of four exons and three introns spanning approx. 1.2 kbp. Two mRNA species are generated from the gene by alternative splicing of the third intron.

Amino Acid Sequence↗
PubMed1981

Primary structure of the Fc region of human immunoglobulin D: implications for evolutionary origin and biological function.

We have determined the complete amino acid sequence of a tryptic Fc delta fragment generated from an intact human IgD (WAH); it is 226 residues long and includes the second (C delta 2) and the third (C delta 3) constant domains of the delta chain. Comparison of the homology of the Fc sequence of the five human immunoglobulin classes suggests that either the delta-chain gene evolved from the alpha-chain gene soon after the divergence of a mu-alpha common ancestor or it evolved from an ancestral gene distinct from both the mu-alpha and the gamma-epsilon common ancestors. Comparative study using a spatial model of the Fc region indicates that the structure of the C delta 3 domain differs extensively from that of the carboxy-terminal domains of other heavy chain classes; this, together with the unique hinge region structure, probably reflects the biological role of IgD as a receptor molecule on the B-lymphocyte surface.

Amino Acid Sequence↗
PubMed1998

EbEST: an automated tool using expressed sequence tags to delineate gene structure.

Large numbers of expressed sequence tags (ESTs) continue to fill public and private databases with partial cDNA sequences. However, using this huge amount of ESTs to facilitate gene finding in genomic sequence imposes a challenge, especially to wet-lab scientists who often have limited computing resources. In an effort to consolidate the information hidden in the vast number of ESTs into a readable and manageable format, we have developed EbEST-a program that automates the process of using ESTs to help delineate gene structure in long stretches of genomic sequence. The EbEST program consists of three functional modules-the first module separates homologous ESTs into clusters and identifies the most informative ESTs within each cluster; the second module uses the informative ESTs to perform gapped alignment and to predict the exon-intron boundary; and the third module generates text file and graphic outputs that illustrate the orientation, exonic structure, and untranslated regions (UTRs) of putative genes in the genomic sequence being analyzed. Evaluation of EbEST with 176 human genes from the ALLSEQ set indicated that it performed in-line with several existing gene finding programs, but was more tolerant to sequencing errors. Furthermore, when EbEST was challenged with query sequences that harbor more than one gene, it suffered only a slight drop in performance, whereas the performance of the other programs evaluated decreased more. EbEST may be used as a stand-alone tool to annotate human genomic sequences with EST-derived gene elements, or can be used in conjunction with computational gene-recognition programs to increase the accuracy of gene prediction. [EbBEST is available at http://EbEST.ifrc.mcw.edu]

Base Sequence↗