A microcomputer interface for a digital audio processor-based data recording system.

An inexpensive interface is described that performs direct transfer of digitized data from the digital audio processor and video cassette recorder based data acquisition system designed by Bezanilla (1985, Biophys. J., 47:437-441) to an IBM PC/XT microcomputer. The FORTRAN callable software that drives this interface is capable of controlling the video cassette recorder and starting data collection immediately after recognition of a segment of previously collected data. This permits piecewise analysis of long intervals of data that would otherwise exceed the memory capability of the microcomputer.     

Relationship between digital information and thermodynamic stability in bacterial genomes

Ever since the introduction of the Watson-Crick model, numerous efforts have been made to fully characterize the digital information content of the DNA. However, it became increasingly evident that variations of DNA configuration also provide an “analog” type of information related to the physicochemical properties of the DNA, such as thermodynamic stability and supercoiling. Hence, the parallel investigation of the digital information contained in the base sequence with associated analog parameters is very important for understanding the coding capacity of the DNA. In this paper, we represented analog information by its thermodynamic stability and compare it with digital information using Shannon and Gibbs entropy measures on the complete genome sequences of several bacteria, including Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Streptomyces coelicolor (S. coelicolor), and Salmonella typhimurium (S. typhimurium). Furthermore, the link to the broader classes of functional gene groups (anabolic and catabolic) is examined. Obtained results demonstrate the couplings between thermodynamic stability and digital sequence organization in the bacterial genomes. In addition, our data suggest a determinative role of the genome-wide distribution of DNA thermodynamic stability in the spatial organization of functional gene groups.     

Applications of video mixing and digital overlay to neuroethology

Neuroethological experiments often require video images of animal behavior and recordings of physiological data to be acquired simultaneously, synchronized with each other, stored, and analyzed together. The use of inexpensive multimedia computers offers new possibilities for mixing video images, analog voltages, and computer data, storing these combined signals to videotape, and extracting quantitative data for analysis. In this paper, we summarize methods for mixing images from multiple video cameras and a Macintosh computer display to facilitate manipulation of data generated during our neurophysiological and behavioral research. These technologies enhance accuracy, speed, and flexibility during experiments, and facilitate selecting and extracting quantitative data from the videotape for further analysis. Three applications are presented: (A) we used an analog video mixer to synchronize neurophysiological recordings with ongoing behaviors of freely moving rats; (B) we used a chroma keyed digital overlay to generate positional data for the rat's face during drinking behavior; and (C) we combined a computer model of a rat's head and whiskers with videos of exploratory behaviors to better track and quantify movements in three dimensions. Although the applications described here are specific to our neuroethological work, these methods will be useful to anyone wishing to combine the signals from multiple video sources into a single image or to extract series of positional or movement data from video frames without frame grabbing.     

A portable system for continuous monitoring of bird nests using digital video recorders

ABSTRACT.   A variety of photographic methods have been described for monitoring nest predation. All have limitations for studying active nests in remote situations, such as size, expense, volume of data recorded, and types of trigger mechanisms. We developed a digital video surveillance system using infrared cameras to monitor predation at bird nests. The main advantage of this system over other video recorders is the small size of the recorder that can run continuously at 29 frames/s for more than 3 days. The recorder's built-in monitor makes it more transportable and allows for easy setup. Digital data is compact, can be reviewed quickly, and requires less physical storage space than videotapes. We recorded nest predation by mammals, birds, and snakes as well as egg and nestling losses not caused by predation. System failure rates were low and the total cost was comparable to ($700 US) video cassette recorders that are often used to monitor nests.     

Why a Constant Number of Vertebrae? Digital Control of Segmental Identity during Vertebrate Development

It is not understood how the numbers and identities of vertebrae are controlled during mammalian development. The remarkable robustness and conservation of segmental numbers may suggest the digital nature of the underlying process. The study proposes a mechanism that allows cells to obtain and store the segmental information in digital form, and to produce a pattern of chromatin accessibility that in turn regulates Hox gene expression specific to the metameric segment. The model requires that a regulatory element be present such that the number of occurrences of the motif between two consecutive Hox genes equals the number of segments under the control of the anterior gene. This is true for the recently discovered hydroxyl radical cleavage 3bp-periodic (HRC3) motif, associated with histone modifications and developmental genes. The finding not only allows the correct prediction of the numbers of segments using only sequence information, but also resolves the 40-year-old enigma of the function of temporal and spatial collinearity of Hox genes. The logic of the mechanism is illustrated in the attached animated video. How different aspects of the proposed mechanism can be tested experimentally is also discussed.     

Biliary excretion of a stretched bilirubin in UGT1A1-deficient (Gunn) and Mrp2-deficient (TR-) rats

The metabolism and biliary excretion of a stretched bilirubin analog with a p-xylyl group replacing the central CH2 hinge were investigated in normal rats, Gunn rats deficient in bilirubin conjugation, and TR- rats deficient in bilirubin glucuronide hepatobiliary transport. Unlike bilirubin, the analog was excreted rapidly in bile unchanged in all three rat strains after intravenous administration. In TR- rats biliary excretion of the analog was diminished, but still substantial, demonstrating that the ATP-binding cassette transporter Mrp2 is not required for its hepatic efflux. These effects are attributable to differences in the preferred conformations of bilirubin and the analog.     

Insertional mutagenesis using a synthetic lac operator

We have developed a novel cassette for generating insertion mutants in multi-copy bacterial plasmids. The cassette consists of synthetic oligodeoxyribonucleotides (oligos) which form a DNA duplex following reconstitution in vitro, due to sequence complementarity. It contains a 21-bp segment of the lac operator (lacZo), to provide a readily detectable phenotypic marker. Bacterial colonies harboring plasmids with insertions of this cassette are blue due to constitutive expression of the lac operon resulting from titration of lac repressor molecules by plasmid-borne lacZo sequences. Synthetic oligos containing a desire sequence may be added to the cassette by complementary ends for targeted insertion into plasmids. Sequencing of the resulting insertion mutants is facilitated by using oligos within the cassette as primers for bidirectional sequencing. This allows a complete characterization of each insertion in terms of location, structure of flanking sequences, and orientation of the inserted oligo. We have used this system to construct a series of mutants in early region 1a genes of human adenovirus type 5. For this purpose we designed a cassette which had all three possible translational reading frames open when inserted in one orientation, and all reading frames closed in the other orientation. The cassette also had BamHI restriction sites at each end which could be used to 'collapse' mutants, reducing the size of each insert to 6 bp.     

Magnetic field exposure and behavioral monitoring system

To maximize the availability and usefulness of a small magnetic field exposure laboratory, we designed a magnetic field exposure system that has been used to test human subjects, caged or confined animals, and cell cultures. The magnetic field exposure system consists of three orthogonal pairs of coils 2 m square x 1 m separation, 1.751 m x 0.875 m separation, and 1.5 m x 0.75 m separation. Each coil consisted of ten turns of insulated 8 gauge stranded copper conductor. Each of the pairs were driven by a constant-current amplifier via digital to analog (D/A) converter. A 9 pole zero-gain active Bessel low-pass filter (1 kHz corner frequency) before the amplifier input attenuated the expected high frequencies generated by the D/A conversion. The magnetic field was monitored with a 3D fluxgate magnetometer (0-3 kHz, +/- 1 mT) through an analog to digital converter. Behavioral monitoring utilized two monochrome video cameras (viewing the coil center vertically and horizontally), both of which could be video recorded and real-time digitally Moving Picture Experts Group (MPEG) encoded to CD-ROM. Human postural sway (standing balance) was monitored with a 3D forceplate mounted on the floor, connected to an analog to digital converter. Lighting was provided by 12 offset overhead dimmable fluorescent track lights and monitored using a digitally connected spectroradiometer. The dc resistance, inductance of each coil pair connected in series were 1.5 m coil (0.27 Omega, 1.2 mH), 1.75 m coil (0.32 Omega, 1.4 mH), and 2 m coil (0.38 Omega, 1.6 mH). The frequency response of the 1.5 m coil set was 500 Hz at +/- 463 microT, 1 kHz at +/- 232 microT, 150 micros rise time from -200 microT(pk) to + 200 microT(pk) (square wave) and is limited by the maximum voltage ( +/- 146 V) of the amplifier (Bessel filter bypassed).     

Importance of secondary structural specificity determinants in protein folding: insertion of a native beta-sheet sequence into an alpha-helical coiled-coil

To examine how a short secondary structural element derived from a native protein folds when in a different protein environment, we inserted an 11-residue beta-sheet segment (cassette) from human immunoglobulin fold, Fab new, into an alpha-helical coiled-coil host protein (cassette holder). This de novo design protein model, the structural cassette mutagenesis (SCM) model, allows us to study protein folding principles involving both short- and long-range interactions that affect secondary structure stability and conformation. In this study, we address whether the insertion of this beta-sheet cassette into the alpha-helical coiled-coil protein would result in conformational change nucleated by the long-range tertiary stabilization of the coiled-coil, therefore overriding the local propensity of the cassette to form beta-sheet, observed in its native immunoglobulin fold. The results showed that not only did the nucleating helices of the coiled-coil on either end of the cassette fail to nucleate the beta-sheet cassette to fold with an alpha-helical conformation, but also the entire chimeric protein became a random coil. We identified two determinants in this cassette that prevented coiled-coil formation: (1) a tandem dipeptide NN motif at the N-terminal of the beta-sheet cassette, and (2) the hydrophilic Ser residue, which would be buried in the hydrophobic core if the coiled-coil structure were to fold. By amino acid substitution of these helix disruptive residues, that is, either the replacement of the NN motif with high helical propensity Ala residues or the substitution of Ser with Leu to enhance hydrophobicity, we were able to convert the random coil chimeric protein into a fully folded alpha-helical coiled-coil. We hypothesized that this NN motif is a "secondary structural specificity determinant" which is very selective for one type of secondary structure and may prevent neighboring residues from adopting an alternate protein fold. These sequences with secondary structural specificity determinants have very strong local propensity to fold into a specific secondary structure and may affect overall protein folding by acting as a folding initiation site.     

A video digitizer for analysis of trunk deformity in scoliosis

Scoliosis is a deformity characterized by lateral curvature of the spine and accompanied by axial rotation of the vertebrae; it often causes varying degrees of trunk deformity. Research has indicated that topographic techniques can be used to describe the disorder and monitor its progression. A video image acquisition system has been designed which reduces the time required to quantify topographic details of the trunk and aids in the diagnosis, monitoring and research of scoliosis. This system integrates the capability of large, expensive grey-scale image acquisition equipment into a small, low-cost diagnostic imaging tool using current technologies and design techniques. The video digitizer accepts a standard NTSC monochromatic video signal as input and the unit is connected to a computer via an EEE-488 bus from which the ditigizer is controlled. The digitizer samples the video signal in real time using a high-speed flash converter controlled by an application-specific integrated circuit; the digital samples are stored in memory until the host computer requests that the information be transferred.     

Stable and strictly controlled expression of LTR-flanked autoregulated expression cassettes upon adenoviral transfer

An autoregulatory bidirectional expression cassette encoding all components necessary for regulated gene expression in a one-step gene transfer was evaluated for use in adenoviral vectors. Adenoviral vectors transducing this cassette provide about 1000-fold regulation. Regulation could be further improved by integrating the cassette as a retroviral vector into the adenoviral backbone. Moreover, with these adeno/retroviral hybrid vectors, the frequency of chromosomal integration is enhanced and about 1% of infected cells show stable chromosomal integration of the autoregulated cassette. In these stably transduced cells high regulation capacity is maintained. To elucidate the molecular mechanism underlying this unexpected observation we investigated the regulation capacity of these cassettes in a viral and non-viral vector background after stable integration into the host's DNA. While naked cassettes show regulated expression that is strongly influenced by the chromosomal surrounding sequences the regulatory capacity of LTR flanked cassettes is highly comparable amongst different cell clones. This strict regulation with little influence from the flanking sequences is obtained when LTR-flanked cassettes are transduced as DNA, by retroviral or by adenoviral infection.     

The digital origin of human language--a synthesis

The fact that all languages known are digital poses the question of their origin. The answer developed here treats language as the interface of information theory and molecular development by showing previously unrecognized isomorphisms between the analog and digital features of language and life at the molecular level. Human language is a special case of signal transduction and hence is subject to the coding aspects of Shannon's theorems and the analog aspects of pattern recognition, each represented by genotype and phenotype. Digital language acquisition is late in evolution and postnatal development and requires a neural reorganization by a mechanism of somatic network programming in response to the environment. Such a mechanism would solve the Chomsky conundrum of how children can learn any language without knowing rules of grammar too numerous to be encoded genotypically.     

Herpes simplex virus type 1/adeno-associated virus hybrid vectors mediate site-specific integration at the adeno-associated virus preintegration site,AAVS1, on human chromosome 19

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors have a large transgene capacity and can efficiently infect many different cell types. One disadvantage of HSV-1 vectors is their instability of transgene expression. By contrast, vectors based on adeno-associated virus (AAV) can either persist in an episomal form or integrate into the host cell genome, thereby supporting long-term gene expression. AAV expresses four rep genes, rep68, -78, -40, and -52. Of those, rep68 or rep78 are sufficient to mediate site-specific integration of the AAV DNA into the host cell genome. The major disadvantage of AAV vectors is the small transgene capacity ( approximately 4.6 kb). In this study, we constructed HSV/AAV hybrid vectors that contained, in addition to the standard HSV-1 amplicon elements, AAV rep68, rep78, both rep68 and -78, or all four rep genes and a reporter gene that was flanked by the AAV inverted terminal repeats (ITRs). Southern blots of Hirt DNA from cells transfected with the hybrid vectors and HSV-1 helper DNA demonstrated that both the AAV elements and the HSV-1 elements were functional in the context of the hybrid vector. All hybrid vectors could be packaged into HSV-1 virions, although those containing rep sequences had lower titers than vectors that did not. Site-specific integration at AAVS1 on human chromosome 19 was directly demonstrated by PCR and sequence analysis of ITR-AAVS1 junctions in hybrid vector-transduced 293 cells. Cell clones that stably expressed the transgene for at least 12 months could easily be isolated without chemical selection. In the majority of these clones, the transgene cassette was integrated at AAVS1, and no sequences outside the ITR cassette, rep in particular, were present as determined by PCR, ITR rescue/replication assays, and Southern analysis. Some of the clones contained random integrations of the transgene cassette alone or together with sequences outside the ITR cassette. These data indicate that the long-term transgene expression observed following transduction with HSV/AAV hybrid vectors is, at least in part, supported by chromosomal integration of the transgene cassette, both randomly and site specifically.     

Blood circulation laboratory investigations with video are less investigative than instructional blood circulation laboratories with live organisms

Live organisms versus digital video of the organisms were used to challenge students' naive ideas and misconceptions about blood, the heart, and circulatory patterns. Three faculty members taught 259 grade 10 biology students in a California high school with students from diverse ethnolinguistic groups who were divided into 5 classes using microscopes (128 students) and 5 classes using digital video (131 students) to compare blood transport among invertebrates, fish, and humans. The "What Is Happening in this Class?" (WIHIC) questionnaire was used for assessment of microscope and video groups to detect students' perception of their learning environment following these teaching interventions. The use of microscopes had a clear effect on the perception of the investigative aspects of the learning environment that was not detected with the video treatment. Findings suggest that video should not replace investigations with live organisms.     

7-Azidoactinomycin D: a novel probe for examining actinomycin D-DNA interactions

The technique of photoaffinity labeling is applied to the actinomycin D system to provide a novel probe for the examination of the interactions of actinomycin D with nucleic acids. The capacity for covalent attachment of actinomycin D will aid greatly in the study of target-site specificities and the correlations of biological effects with biophysical DNA interactions. Through chemical modification of the parent actinomycin D molecule with a photoreactive azido substituent, a functional analog of the parent actinomycin D is generated having equilibrium binding properties identical to those of the parent molecule yet with the capacity to form a covalent attachment to DNA upon photolysis. The results presented here describe the noncovalent interactions of this photoreactive probe to DNA (absence of light) and compares the binding properties observed to those of the parent actinomycin D and 7-aminoactinomycin D analog. These studies demonstrate that the DNA binding properties (i.e. binding affinity, binding site size, and sequence specificity) retained by the 7-azidoactinomycin D, thus providing a suitable probe for examining actinomycin D-DNA interactions.     

Dimensional measurements on images from a video camera or cassette recorder using a BBC microcomputer

We have developed a system that enables a BBC microcomputer to make dimensional measurements on images from a video camera or cassette recorder without a framestore. The RGB output of the computer is synchronized to the output of the camera or recorder and the two displayed simultaneously on the same monitor. Measurements can be made directly from the displayed image using points indicated by the computer graphics cursor via its keyboard. Distances can be measured with a precision of about 0.4%. The system is very versatile but is especially useful for measuring the freeze-frame output from a Philips standard videotape.     

Transfer of a genetic marker from a megaplasmid of Anabaena sp. strain PCC 7120 to a megaplasmid of a different Anabaena strain.

The 410-kb alpha megaplasmid of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was found to bear the nucA gene that encodes a sugar-nonspecific nuclease. That gene was mutated by insertion of a cassette that confers resistance to neomycin. The resulting strain, AMP2, was mated with a streptomycin-resistant derivative of Anabaena sp. strain PCC 7118, a strain that does not form heterocysts. Cells resistant to both neomycin and streptomycin that were derived from such matings were found to bear the neomycin resistance cassette of the donor strain in a larger megaplasmid characteristic of the recipient strain and did not form heterocysts. This is the first example of transfer of a genetic marker directly between strains of cyanobacteria in which incontrovertible physical evidence of transfer has been obtained. DNA sequences homologous to the nucA gene were present in 13 heterocyst-forming cyanobacteria that were tested but in none of six diverse unicellular strains that were examined.     

15种氨基酸随机肽库的小盒式DNA编码文库的构建

采用小盒式DNA编码文库的构建策略,选取在进化上可能起源较早的15种氨基酸,按照其简并密码子合成了一个为10个随机氨基酸编码的小盒式DNA模板,经过连续3轮的PCR扩增、酶切及连接的小盒式文库组装过程,成功构建了一个文库容量达1.31×10¹²/ml,随机编码区长达97个氨基酸的小盒式DNA编码文库。