Locating a site on the maize B chromosome that controls preferential fertilization.

In maize, the B chromosome can undergo nondisjunction at the second pollen mitosis, producing sperm with two B chromosomes and sperm with zero B chromosomes. Preferential fertilization is the ability of the sperm carrying two B chromosomes to transmit more frequently to the embryo of a kernel than the sperm lacking the B chromosome. A translocation involving the B chromosome and chromosome 9, TB-9Sb, has been used to study preferential fertilization. The B-9 chromosome has the same properties of nondisjunction and preferential fertilization as the standard B chromosome. Deletion derivatives of B-9, which lack the centric heterochromatin and possibly some adjacent euchromatin, were tested for their ability to induce preferential fertilization. They were found to lack the capacity for preferential fertilization.     

Attachment site mutants of bacteriophage lambda

Insertion and excision of the chromosome of phage λ occurs by recombination at special regions of the phage and bacterial chromosomes known as attachment sites (alt's). We have isolated att mutants which display reduced recombination frequencies. The mutations are cis-dominant, trans-recessive, and can be crossed into a phage, bacterial or prophage att. These results suggest that the att's, although different over-all, include the same DNA sequence as part of their structure, and that the mutations reside in these sequences. Crosses between mutant and wild-type att's occasionally yield heterozygotes. This result suggests that recombination of the att's generates complementary single-strands via staggered nicks in these common sequences. Recombinant att's are then formed by the interannealing of single-strands of different att's followed by ligation.     

Stochastic Properties of Discrete Waves of the Limulus Photoreceptor

In the dark-adapted photoreceptor of the horseshoe crab, Limulus, transient discrete depolarizations of the cell membrane, discrete waves, occur in total darkness and their rate of occurrence is increased by illumination. The individual latencies of the discrete waves evoked by a light stimulus often cannot be resolved because the discrete waves overlap in time. The latency of the first discrete wave that follows a stimulus can be determined with reasonable accuracy. We propose a model which allows us to make an estimate of the distribution of the latencies of the individual light-evoked discrete waves, and to predict the latency distribution of the first discrete wave that follows a stimulus of arbitrary intensity-time course from the latency distribution of the first discrete wave that follows a brief flash of light. For low intensity stimuli, the predictions agree well with the observations. We define a response as the occurrence of one or more discrete waves following a stimulus. The distribution of the peak amplitudes of responses suggests that the peak amplitude of individual discrete waves sometimes has a bimodal distribution. The latencies of the two types of discrete waves, however, follow similar distributions. The area under the voltage-time curve of responses that follow equal energy long (1.25 sec) and short (10 msec) light stimuli follows similar distributions, and this suggests that discrete waves summate linearly.     

Specificity determinants for bacteriophage lambda DNA replication: I. A chain of interactions that controls the initiation of replication

The genetic elements which control autonomous DNA replication differ in functional specificity among coliphage λ, the coliphages φ80 and 82, and the Salmonella phage P22. Hybrid phages derived by genetic recombination between λ and each of these related phages have been used to define and to localize specificity determinants for DNA replication.In λ-P22 hybrid phages (Hilliker & Botstein, 1976) the replication control elements segregate as an intact unit. By contrast, some viable λ-φ80 and λ.82 hybrid phages arise by recombination within the replication control region, in a small interval inside structural gene O. From the properties of such hybrid phages, we infer that the O gene product of λ and the functionally equivalent proteins of φ80 and 82 each interact with a specific nucleotide sequence in the cognate ori site, the DNA target for control of the origin of replication. With respect to this interaction, both the O products and the receptor sequences within ori show stringent type specificity. The donor and receptor specificity determinants for the ori-O interaction lie within an interval of less than 400 base-pairs.The O gene product also interacts with the product of replication gene P (Tomizawa, 1971). The O-P interaction displays limited type specificity; the P-like protein of φ80 can function together with the O protein of λ, but the P protein of λ cannot function with the O-like protein of φ80. The specificity determinants for the O-P interaction can be separated from those for the ori-O interaction.We propose that a chain of interactions between ori, O product, P product, and replication functions of the bacterial host, Escherichia coli, controls specific template selection and the assembly of the essential replication apparatus in the initiation of λ DNA replication.     

A conformational switch controls the DNA cleavage activity of lambda integrase

The bacteriophage lambda integrase protein (lambda Int) belongs to a family of tyrosine recombinases that catalyze DNA rearrangements. We have determined a crystal structure of lambda Int complexed with a cleaved DNA substrate through a covalent phosphotyrosine bond. In comparison to an earlier unliganded structure, we observe a drastic conformational change in DNA-bound lambda Int that brings Tyr342 into the active site for cleavage of the DNA in cis. A flexible linker connects the central and the catalytic domains, allowing the protein to encircle the DNA. Binding specificity is achieved through direct interactions with the DNA and indirect readout of the flexibility of the att site. The conformational switch that activates lambda Int for DNA cleavage exposes the C-terminal 8 residues for interactions with a neighboring Int molecule. The protein interactions mediated by lambda Int's C-terminal tail offer a mechanism for the allosteric control of cleavage activity in higher order lambda Int complexes.     

Electrophysiological Properties of Cells in the Median Ocellus of Limulus

Two types of photoreceptors are found in the median ocellus of Limulus. One type is maximally sensitive to ultraviolet (UV) light, the other to green light; they are called UV and VIS cells, respectively. Biphasic receptor potentials, consisting of a small initial hyperpolarizing phase and a later slow depolarizing phase, can be recorded from both receptor types. These biphasic responses are elicited in UV cells in response to long-wavelength light, and in VIS cells in response to ultraviolet light. Another type of hyperpolarizing response can be recorded in UV cells: after a bright ultraviolet stimulus, the cell remains depolarized; long-wavelength light rapidly returns the membrane potential to its value preceding ultraviolet illumination (this long-wavelength-induced potential change is called a "repolarizing response"). Also, a long-wavelength stimulus superimposed during a UV stimulus elicits a sustained repolarizing response. A third cell type (arhabdomeric cell) found in the median ocellus generates large action potentials and is maximally sensitive to UV light. Biphasic responses and repolarizing responses also can be recorded from arhabdomeric cells. The retina is divided into groups of cells; both UV cells and VIS cells can occur in the same group. UV cells in the same group are electrically coupled to one another and to an arhabdomeric cell.     

Duplication of the bacteriophage lambda cohesive end site: genetic studies

A derivative of bacteriophage λ has been isolated that contains a duplication of the cohesive end site. To support this conclusion, the duplicated region has bean recovered by segregation from a lysogen of the duplication strain, and a derivative of the duplication strain was constructed that is heterozygous for the λ genes R and A, which bracket the cohesive end site. Duplication strains show no instability during lysogenization, suggesting that the virus particles each contain a single DNA molecule. During lytic growth, however, the strain is unstable and the duplication is frequently lost, even in the absence of all known recombination systems. Loss of the duplication is ascribed to cleavage of both cohesive end sites by the chromosome maturation system. Thus both cohesive end sites are functional, i.e. capable of being cleaved. No transfer of the duplicated region occurs in the absence of the known recombination systems. Thus, during λ chromosome maturation, cleavage of DNA molecules occurs but rejoining of cleaved molecules does not.     

Bacteriophage lambda derivatives carrying two copies of the cohesive end site

A spontaneously arising tandem duplication derivative of bacteriophage lambda has been isolated, which carries two copies of the site where the cohesive ends are formed (designated cos). Its structure has been determined by electron microscopy of DNA heteroduplexes. These heteroduplexes reveal that the duplication is usually, but not always, carried on the left end of the chromosome. A second duplication phage having two copies of cos, constructed by Feiss &; Campbell (1974), has also been studied by electron microscopy and is found to have a similar property.Unlike most tandem duplication derivatives of phage λ, the mutant studied here is not stable during growth in the absence of generalized recombination, but segregates both the triplication and the parental phage. This verifies that both cos sites are functional. The triplication does not arise as a result of end-to-end aggregation of phage chromosomes or site-specific recombination catalyzed by the chromosome maturation system at cos. It must therefore result from the cutting of mature ι chromosomes from concatemeric replication intermediates. The pattern of cutting observed shows that the λ cohesive ends are not created by a free nuclease acting on unpackaged DNA. The cutting appears to be influenced by the amount of DNA previously packaged into a phage head. A model for λ packaging is presented which explains the results.The duplication phage of Feiss &; Campbell (1974) carries a novel addition containing self-complementary sequences.     

Effect of phospholipid removal on the kinetics of the metarhodopsin I to metarhodopsin II reaction.

The kinetics of the metarhodopsin (meta) I → metarhodopsin II reaction have been studied by flash photolysis in two different types of preparations of bovine rhodopsin: (i) digitonin-solubilized rod outer segment (ROS) membranes with a molar ratio of phospholipid to rhodopsin of approximately 90, and (ii) digitonin-solubilized phospholipid-free rhodopsin with a molar ratio of phospholipid to rhodopsin of less than 0.2. At 20 °C the kinetics in both preparations are multiexponential, but four terms are required to fit the data with the solubilized membranes, whereas only two are required with the phospholipid-free preparation. Thus, phospholipid removal simplifies the kinetics of the meta I → meta II reaction, but the resulting preparation still does not show first-order kinetics. The ratio of the time constants of these two components with detergent-solubilized phospholipid-free rhodopsin was nearly equal to the values found with ROS particles, rhodopsin-phospholipid recombinants and intact rabbit eyes. This suggests a common origin for these two components in all these preparations and appears to exclude heterogeneity in bound phospholipid as the basis of these two-component kinetics.     

Mapping of restriction sites in the attachment site region of bacteriophage lambda

Summary A fine structure map of theEcoRI fragment containing the lambda attachment-site region has been constructed. 38 different restriction endonucleases have been employed and 170 sites located in this fragment. In addition, sites in adjacent regions have been determined for several enzymes. Complete cleavage maps of the entire lambda genome have been obtained for endonucleasesBglII,BluI,KpnI,SacI,SacII,SalI andXbaI. The strategy employed for mapping included comparison of deletion and substitution mutants, analysis of mixed digests, and detailed analysis of subfragments.     

Bending and supercoiling of DNA at the attachment site of bacteriophage lambda

Integration of the DNA of bacteriophage lambda into the chromosome of E. coli depends on the formation of a complex nucleoprotein array at a specific locus on the phage genome, the attachment site. Recent work shows how bending of this DNA (induced by a specific DNA-binding protein), and strain in this DNA (induced by supercoiling) contribute to the formation of the nucleoprotein structure. Further, there are new insights into the way this structure directs critical events during recombination.     

Architecture of the 99 bp DNA-six-protein regulatory complex of the lambda att site

The highly directional and tightly regulated recombination reaction used to site-specifically excise the bacteriophage lambda chromosome out of its E. coli host chromosome requires the binding of six sequence-specific proteins to a 99 bp segment of the phage att site. To gain structural insights into this recombination pathway, we measured 27 FRET distances between eight points on the 99 bp regulatory DNA bound with all six proteins. Triangulation of these distances using a metric matrix distance-geometry algorithm provided coordinates for these eight points. The resulting path for the protein-bound regulatory DNA, which fits well with the genetics, biochemistry, and X-ray crystal structures describing the individual proteins and their interactions with DNA, provides a new structural perspective into the molecular mechanism and regulation of the recombination reaction and illustrates a design by which different families of higher-order complexes can be assembled from different numbers and combinations of the same few proteins.     

178-Nucleotide sequence surrounding the cos site of bacteriophage lambda DNA.

A nucleotide sequence of 61 nucleotides at the left end and 117 nucleotides at the right end of DNA from bacteriophage lambdacI857Sam7 was determined by the Maxam and Gilbert method. A perfect inverted repeat sequence of 10 nucleotides is near the left end, and one of 15 nucleotides is near the right end. DNA from another closely related lambda strain, lambdacI857prm116Sam7, has about 10% divergence in the sequence of the first 110 nucleotides at the right end and has a 17-member perfect inverted repeat sequence.     

Signaling states of rhodopsin. Formation of the storage form,metarhodopsin III,from active metarhodopsin II

Vertebrate rhodopsin consists of the apoprotein opsin and the chromophore 11-cis-retinal covalently linked via a protonated Schiff base. Upon photoisomerization of the chromophore to all-trans-retinal, the retinylidene linkage hydrolyzes, and all-trans-retinal dissociates from opsin. The pigment is eventually restored by recombining with enzymatically produced 11-cis-retinal. All-trans-retinal release occurs in parallel with decay of the active form, metarhodopsin (Meta) II, in which the original Schiff base is intact but deprotonated. The intermediates formed during Meta II decay include Meta III, with the original Schiff base reprotonated, and Meta III-like pseudo-photoproducts. Using an intrinsic fluorescence assay, Fourier transform infrared spectroscopy, and UV-visible spectroscopy, we investigated Meta II decay in native rod disk membranes. Up to 40% of Meta III is formed without changes in the intrinsic Trp fluorescence and thus without all-trans-retinal release. NADPH, a cofactor for the reduction of all-trans-retinal to all-trans-retinol, does not accelerate Meta II decay nor does it change the amount of Meta III formed. However, Meta III can be photoconverted back to the Meta II signaling state. The data are described by two quasi-irreversible pathways, leading in parallel into Meta III or into release of all-trans-retinal. Therefore, Meta III could be a form of rhodopsin that is stored away, thus regulating photoreceptor regeneration.     

Conformation analysis of a surface loop that controls active site access in the GH11 xylanase A from Bacillus subtilis

Xylanases (EC 3.2.1.8 endo-1,4-glycosyl hydrolase) catalyze the hydrolysis of xylan, an abundant hemicellulose of plant cell walls. Access to the catalytic site of GH11 xylanases is regulated by movement of a short β-hairpin, the so-called thumb region, which can adopt open or closed conformations. A crystallographic study has shown that the D11F/R122D mutant of the GH11 xylanase A from Bacillus subtilis (BsXA) displays a stable “open” conformation, and here we report a molecular dynamics simulation study comparing this mutant with the native enzyme over a range of temperatures. The mutant open conformation was stable at 300 and 328 K, however it showed a transition to the closed state at 338 K. Analysis of dihedral angles identified thumb region residues Y113 and T123 as key hinge points which determine the open-closed transition at 338 K. Although the D11F/R122D mutations result in a reduction in local inter-intramolecular hydrogen bonding, the global energies of the open and closed conformations in the native enzyme are equivalent, suggesting that the two conformations are equally accessible. These results indicate that the thumb region shows a broader degree of energetically permissible conformations which regulate the access to the active site region. The R122D mutation contributes to the stability of the open conformation, but is not essential for thumb dynamics, i.e., the wild type enzyme can also adapt to the open conformation.     

Properties of visual cells in the lateral eye of Limulus in situ.

Excitatory properties of visual cells in the lateral eye of Limulus, investigated by optic nerve recordings in situ, differ significantly from the properties of cells in the classical, excised eye preparation. The differences suggest the possibility that two receptor mechanisms function in the eye in situ: one mechanism encodes low light intensities and the other responds to high intensities. The two mechanisms enable each ommatidium to respond over an intensity range of approximately 10 log units. This hypothesis was tested by measuring the increment threshold and the spectral sensitivity, by studying light and dark adaptation, and by analyzing the variability of the impulse discharge. Although the results do not conclusively identify two receptor mechanisms, they indicate that a process or a part of a process that functions in the eye in situ is abolished by excising the eye or cutting off its blood supply.