A pulse generator simulating slit-scan chromosome analysis signals

A simple circuit is described for generating a variety of electronic pulses to test hardware and software for slit-scan chromosome analysis in a flow cytometer. The pulse shape can be changed to have different numbers of local minima, thereby simulating fluorescence pulses from acrocentric, monocentric, and dicentric chromosomes. Long pulses simulate aggregates of chromosomes. The pulse repetition rate as well as the pulse amplitude is variable. Although the circuitry is built with only three integrated circuits, the pulse-to-pulse variation in shape and height is quite small. After digitization of the analog signals, the constructed histograms of pulse integrals show a relative coefficient of variation below 1%. This signal generator provides a valuable tool for a number of electronic test applications that would otherwise require expensive standard particles analyzed in a well-tuned flow cytometer.     

Computer-controlled pulse modulation system for analysis of photoacoustic signals in the time domain

A newly developed photoacoustic system for measurement of photosynthetic reactions in intact leaves is described. The system is based on pulsed light-emitting diodes, the pulse program and pulse response analysis being computer controlled. Separation of various components in the overall photoacoustic signal is achieved by curve fitting analysis of the responses following individual measuring light pulses in the millisecond time domain. This procedure is in distinction to the conventionally used analysis in the frequency domain, with the advantage that various signal components are obtained by on-line deconvolution, yielding simultaneous recordings of photothermal (complement of energy storage) and photobaric (evolution and uptake) signals. The basic components of the new system are described by block diagrams and the principal steps for deconvolution of the overall photoacoustic response are outlined. An example of application with simultaneous recording of chlorophyll fluorescence is given. It is apparent that the photobaric uptake component represents a significant part of the overall signal, particularly during induction of photosynthesis after dark-adaptation. This component probably contains not only O₂-uptake but uptake of CO₂ as well.Abbreviations PA photoacoustic - LED light-emitting-diode - RAM random access memory     

The elucidation of protein function by sequence motif analysis

Protein sequence motifs are acquiring increasing prominencein the area of sequence analysis. This review describes thecurrent methods of their construction and their use in the determinationof protein function, and offers guidelines on interpreting dataobtained. An appendix is attached which refers to 200 motifsof various kinds.     

The ASTRAL compendium for protein structure and sequence analysis

The ASTRAL compendium provides several databases and tools to aid in the analysis of protein structures, particularly through the use of their sequences. The SPACI scores included in the system summarize the overall characteristics of a protein structure. A structural alignments database indicates residue equivalencies in superimposed protein domain structures. The PDB sequence-map files provide a linkage between the amino acid sequence of the molecule studied (SEQRES records in a database entry) and the sequence of the atoms experimentally observed in the structure (ATOM records). These maps are combined with information in the SCOPdatabase to provide sequences of protein domains. Selected subsets of the domain database, with varying degrees of similarity measured in several different ways, are also available. ASTRALmay be accessed at http://astral.stanford.edu/     

Alkylation of cysteine with acrylamide for protein sequence analysis.

Alkylation of cysteine in proteins with acrylamide under mildly alkaline conditions yields a thioether derivative, Cys-S-beta-propionamide (Cys-S-Pam), which is stable during automated Edman degradation. Its phenylthiohydantoin derivative, PTH-Cys-S-Pam, is easily separated from other PTH-amino acids by HPLC and is thus useful for cysteine identification during protein sequencing. PTH-Cys-S-Pam was first noticed during sequencing polypeptides blotted onto polyvinylidene difluoride membranes from polyacrylamide gels, in which cysteine had reacted with residual unpolymerized acrylamide. Cysteine in proteins is easily alkylated by reaction of proteins in aqueous solution with acrylamide. Methods are also presented for alkylation of cysteine in proteins adsorbed on fiberglass disks in the reaction cartridge of a protein sequencer. Finally, PTH-Cys-S-Pam was synthesized chemically. The synthetic compound is unstable in neutral solution, but can be stabilized by acidification. It has the same HPLC retention time as the product formed from cysteine when sequencing proteins alkylated with acrylamide.     

Designing hardware for protein sequence analysis

We present the architecture of PROSIDIS, a special purpose co-processor designed to search for the occurrence of substrings similar to a given 'template string' within a proteome. Actual tests show speed up figures ranging from 5 to 50 with respect to conventional general-purpose processors. AVAILABILITY: the PROSIDIS configuration file and the c code are available at http://www.enea.it/hpcn/php/rosato/     

Degradation signals in the lysine-asparagine sequence space.

The N-degrons, a set of degradation signals recognized by the N-end rule pathway, comprise a protein's destabilizing N-terminal residue and an internal lysine residue. We show that the strength of an N-degron can be markedly increased, without loss of specificity, through the addition of lysine residues. A nearly exhaustive screen was carried out for N-degrons in the lysine (K)-asparagine (N) sequence space of the 14-residue peptides containing either K or N (16 384 different sequences). Of these sequences, 68 were found to function as N-degrons, and three of them were at least as active and specific as any of the previously known N-degrons. All 68 K/N-based N-degrons lacked the lysine at position 2, and all three of the strongest N-degrons contained lysines at positions 3 and 15. The results support a model of the targeting mechanism in which the binding of the E3-E2 complex to the substrate's destabilizing N-terminal residue is followed by a stochastic search for a sterically suitable lysine residue. Our strategy of screening a small library that encompasses the entire sequence space of two amino acids should be of use in many settings, including studies of protein targeting and folding.     

Principal components analysis of protein sequence clusters

Sequence analysis of large protein families can produce sub-clusters even within the same family. In some cases, it is of interest to know precisely which amino acid position variations are most responsible for driving separation into sub-clusters. In large protein families composed of large proteins, it can be quite challenging to assign the relative importance to specific amino acid positions. Principal components analysis (PCA) is ideal for such a task, since the problem is posed in a large variable space, i.e. the number of amino acids that make up the protein sequence, and PCA is powerful at reducing the dimensionality of complex problems by projecting the data into an eigenspace that represents the directions of greatest variation. However, PCA of aligned protein sequence families is complicated by the fact that protein sequences are traditionally represented by single letter alphabetic codes, whereas PCA of protein sequence families requires conversion of sequence information into a numerical representation. Here, we introduce a new amino acid sequence conversion algorithm optimized for PCA data input. The method is demonstrated using a small artificial dataset to illustrate the characteristics and performance of the algorithm, as well as a small protein sequence family consisting of nine members, COG2263, and finally with a large protein sequence family, Pfam04237, which contains more than 1,800 sequences that group into two sub-clusters.     

The effect of protein depletion on the rate of protein synthesis in rat liver.

In order to understand the mechanism of decreased protein synthesis in the liver of rats fed a protein-free diet, the average polypeptide chain assembly time (tc) was measured by the method of Mathews et al. (J. Biol. Chem. (1973) 248, 1329). For rats fed a normal diet, tc in liver in vivo was 1.28 min. A 10-day period of protein depletion led to a value of tc = 2.08 min, corresponding to a 38% depression in polypeptide elongation rate. Protein depletion caused an extensive breakdown of hepatic polysomes and refeeding of a complete mixture of amino acids resulted in rapid recovery of polysomal profile. But tc in the liver of the refed animals gave still depressed value of 1.95 min. The amount and size distribution of poly(A)-containing mRNA in the liver, as determined by [3H]poly(U) hybridization, were the same for normal and depleted groups. These results suggest that both initiation and elongation steps of protein synthesis are depressed in the liver of protein-depleted rats. Refeeding of amino acid mixture rapidly restores initiation but not elongation activity.     

Correction of the cDNA-derived protein sequence of prostatic spermine binding protein: pivotal role of tandem mass spectrometry in sequence analysis

Spermine binding protein (SBP) is a rat ventral prostate protein that binds various polyamines, and the level of this protein and its mRNA is regulated by androgens. Previously, the cDNA for SBP was cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from partial amino acid sequencing of the purified protein were consistent with the amino acid sequence deduced from the cDNA. However, the amino terminus of the protein was blocked, and therefore, direct protein sequence information confirming the cDNA reading frame of this region could not be obtained by Edman degradation. We have now employed an integrated approach using fast atom bombardment mass spectrometry, tandem mass spectrometry, and conventional sequencing methodologies to establish the amino-terminal sequence of the protein and to identify an amino acid sequence (35 residues) present in the purified protein but missing from the amino acid sequence deduced from cDNA clones for this protein. The missing piece of cDNA corresponds to an exon found in mouse genomic clones for a protein similar to rat SBP. Therefore, the cDNA clones for rat SBP may represent splicing variants that lack the sequence information of one exon. The blocked amino terminus of the protein was identified as 5-oxopyrrolidine-2-carboxylic acid. Mass spectrometry also provided evidence regarding glycosylation of the protein. The first of two potential glycosylation sites clearly carries carbohydrate; the second site is, at most, only partially glycosylated.     

New tools for protein sequence analysis.

Analysis of protein sequence is an important tool in studies of both native and recombinant proteins. Novel techniques and instrumentation which facilitate determination of protein primary structure have recently been developed.     

Human rhinovirus 2: complete nucleotide sequence and proteolytic processing signals in the capsid protein region.

cDNA clones representing the entire genome of human rhinovirus 2 have been obtained and used to determine the complete nucleotide sequence. The genome consists of 7102 nucleotides and possesses a long open reading frame of 6450 nucleotides; this reading frame is initiated 611 nucleotides from the 5' end and stops 42 nucleotides from the polyA tract. The N-terminal sequences of three of the viral capsid proteins have been elucidated, thus defining the positions of three cleavage sites on the polyprotein. The extensive amino acid sequence homology with poliovirus and human rhinovirus 14 enabled the other cleavage sites to be predicted. Cleavages in the 3' half of the molecule appear to take place predominantly at Gln-Gly pairs, whereas those in the 5' half (including the capsid proteins) are more heterogeneous.     

Downstream sequence elements with different affinities for the hnRNP H/H' protein influence the processing efficiency of mammalian polyadenylation signals

Auxiliary factors likely play an important role in determining the polyadenylation efficiency of mammalian pre-mRNAs. We previously identified an auxiliary factor, hnRNP H/H′, which stimulates 3′-end processing through an interaction with sequences downstream of the core elements of the SV40 late polyadenylation signal. Using in vitro reconstitution assays we have demonstrated that hnRNP H/H′ can stimulate processing of two additional model polyadenylation signals by binding at similar relative downstream locations but with significantly different affinities. A short tract of G residues was determined to be a common property of all three hnRNP H/H′ binding sites. A survey of mammalian polyadenylation signals identified potential G-rich hnRNP H/H′ binding sites at similar downstream locations in ~34% of these signals. All of the novel G-rich elements tested were found to bind hnRNP H/H′ protein and the processing of selected signals identified in the survey was stimulated by the protein both in vivo and in vitro. Downstream G-rich tracts, therefore, are a common auxiliary element in mammalian polyadenylation signals. Sequences capable of binding hnRNP H protein with varying affinities may play a role in determining the processing efficiency of a significant number of mammalian polyadenylation signals.