Gene assignment by means of somatic cell hybrids: a new method for the analysis of data

Summary A statistical approach to the interpretation of data from gene assignment with somatic cell hybrids is presented. The observed data are analysed under a variety of hypotheses. The fit to the hypotheses is compared by means of the likelihood obtained under a given hypothesis. Two of these hypotheses are related to fundamental questions: is a gene responsible for the enzyme observation and if so, is that gene located on a specific chromosome or could it change its position and be sometimes on chromosome j and, in another hybrid line, on chromosome k? The other hypotheses concern the assignment of the gene to just one of the chromosomes.To improve the traditional data analysis approach we considered additional information: the uncertainties and possible errors of laboratory methods in all our calculations and the length of the donor chromosomes in connection with one specific hypothesis.This method allows us to account for the reliability of the investigation methods and the nature of the hybrid lines involved. Data can be evaluated at different error probabilities within a realistic range in order to compare and discuss results.     

Test of circular dichroism (CD) methods for crambin and CD-assisted secondary structure prediction of its homologous toxins

Methods that analyze protein circular dichroism (CD) spectra for fractions of secondary structure are evaluated for the plant protein crambin, which has a known high-resolution crystal structure. In addition, a two-step secondary structure prediction scheme is presented and used for the toxins homologous to crambin, shown by others to have secondary structures similar to crambin. The test of CD spectral analysis methods with the protein crambin employed two computer programs and several CD basis sets. Crambin's crystal structure, known to 0.945A resolution (Hendrickson, W.A., Teeter, M.M. Nature 290:107-113, 1981), allows accurate evaluation of results. Analysis with the protein spectra basis sets (Provencher, S.W., Gl?ckner, J. Biochemistry 20:33-37, 1981) as modified (Manavalan, P., Johnson, W.C., Jr. Anal. Biochem. 167:76-85, 1987) agreed most closely with crambin's crystal structure. This method was then applied to the CD spectra of the membrane-active toxins homologous to crambin (alpha 1- and beta-purothionin, phoratoxin A and B, and viscotoxin A3 and B). The new program SEQ (pronounced "seek") was developed to assign the secondary structure along the protein chain in a hierarchical fashion and applied to the plant toxins. The method constrained the secondary structure fractions to those from CD analysis and combined standard statistical methods with amphipathic helix location. Both CD-arrived secondary structure percentages and sequence assignment indicate that the viscotoxins are structurally most similar to crambin. Purothionin's secondary structure was predicted to be fundamentally similar to crambin's with a difference at the start of the first helix. This assignment agreed with Raman and NMR analyses of purothionin and lends validity to the method presented here. Differences from the NMR in the CD secondary structure fraction analysis for phoratoxin suggest interference in the CD from tryptophan residues.     

1H NMR study of the interaction of bacteriophage λ Cro protein with the O R 3 operator

The 17 base pair operator O _R ³ oligonucleotide, which is the preferential binding site for the Cro repressor of phage , was studied by two-dimensional NMR spectroscopy. A sequential assignment procedure based on two-dimensional Nuclear Overhauser Effect (NOESY) and scalar coupling correlated (COSY) NMR spectroscopy, together with the knowledge of the oligodesoxynucleotide sequence, made it possible to assign the non-exhangeable base protons and the H1 and the H2-H2 sugar protons of the O _R ³ operator DNA. The pattern of the observed NOE connectivities is consistent with a right-handed helical DNA structure. The base and sugar proton assignments provide the necessary information for further studies of the O _R ³ operator — Cro repressor interaction.Abbreviations COSY correlated spectroscopy - FID free induction decay - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect spectroscopy - RD relaxation delay - TSP sodium 3-trimethylsilyl-(2,2,3,3-²H₄)propionate - EDTA sodium ethylendiamine tetraacetate     

1H and 15N resonance assignments and secondary structure of the carbon monoxide complex of sperm whale myoglobin

Summary Sequence-specific backbone ¹H and ¹⁵N resonance assignments have been made for 95% of the amino acids in sperm whale myoglobin, complexed with carbon monoxide (MbCO). Many assignments for side-chain resonances have also been obtained. Assignments were made by analysis of an extensive series of homonuclear 2D spectra, measured with unlabeled protein, and both 2D and 3D ¹H-¹⁵N-correlated spectra obtained from uniformly ¹⁵N-labeled myoglobin. Patterns of medium-range NOE connectivities indicate the presence of eight helices in positions that are very similar to those found in the crystal structures of sperm whale myoglobin. The resonance assignments of MbCO form the basis for determination of the solution structure and for hydrogen-exchange measurements to probe the stability and folding pathways of myoglobin. They will also form a basis for assignment of the spectra of single-site mutants with altered ligand-binding properties.     

Sequential1H-NMR assignments of neurotoxin III from the sea anemoneHeteractis macrodactylus and structural comparison with related toxins

The complete sequence-specific assignment of resonances in the¹H-NMR spectrum of the polypeptide neurotoxin III (Hm III) from the sea anemoneHeteractis macrodactylus is described. Comparison of the chemical shifts and pattern of NOEs for Hm III with those for the related toxin Hp III fromHeteractis paumotensis, which differs only in the substitution of Asn for Tyr at position 11, shows that the overall secondary and tertiary structures are conserved. The largest differences in chemical shift caused by the substitution at position 11 are observed for the NH resonances of Arg-13, Thr-14, Ala-15, Leu-17, and Cys-26. The CH resonances influenced most are those of ASP-6, Gly-9, Leu-17, and Glu-42, while the most affected CH resonances are from Leu-17, Glu-28, and Lys-32. The absence of long-range NOEs to the aromatic ring of Tyr-11 as well as the lack of significant chemical shift effects on residues outside the loop comprising residues 7–16 confirm that this part of the loop makes no long-lived contacts with the rest of the molecule. The deviations from random coil shifts of Hm III are compared with those of the related anemone toxins Hp II, Hp III, and toxin I fromStichodactyla helianthus (Sh I). The similarity in deviations in chemical shift as a function of sequence position for these four toxins emphasizes the overall structural homology among these polypeptides.     

Monogamy in black vultures: genetic evidence from DNA fingerprinting

Recent research has indicated that avian mating systems maycommonly deviate from monogamy due to extrapair fertilizations(EPFs). Because the majority of avian species have long beenconsidered monogamous, accurate measurement of the frequencyof EPFs in a variety of species is important to enhance understandingof the evolution of avian mating systems. We used DNA fingerprintingto investigate the apparently monogamous mating system of blackvultures (Coragyps airaius) by assaying parentage within severalnuclear families. Monogamy is suggested in black vultures becausemated pairs exhibit long-term pair bonding and year-round association,and share incubation and nestling feeding duties equally. Thirtytwobreeders and 36 nestlings representing 16 complete nuclear familieswere tagged for individual identification and sampled for DNAanalysis using 2 restriction enzymes and 3 probes for hypervariableregions. Putative parents were assigned parentage in all cases.We empirically examined the probability of detecting EPFs bycomparing nestlings' fingerprints to those of a putative parentand another randomly chosen adult. All putative parents couldbe assigned with 95%confidence and all outside adults couldbe similarly excluded. There is therefore no evidence for successfulEPFs in this population, indicating a mating system that doesnot deviate from strict monogamy. The complex social behaviorof black vultures may eliminate the opportunity for EPFs dueto the prohibition of copulations in the presence of relatives.     

A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins

Summary We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of ¹³C/¹⁵N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (¹H and ¹³C) and backbone (¹H, ¹³C and ¹⁵N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.     

Spatial autocorrelation invalidates the Dow-Cheverud test

Dow and Cheverud (Am. J. Phys. Anthropol. 68:367–373, 1985) have published a statistical test for comparing any three similarity matrices. Using both simulations and analytical arguments, I establish that the presence of spatial autocorrelation, a common feature of geographically based anthropological and biological data, causes this test to reject too often. Increasing the spatial autocorrelation increases the spurious rejection rate. About 20% of the papers that reference Dow and Cheverud's paper have used their test with spatially autocorrelated data. Mantel's (Cancer Res. 27:209–220, 1967) method, when used as a test of spatial autocorrelation, is unaffected by these considerations, since its null hypothesis is that the data are uncorrelated. © 1992 Wiley-Liss, Inc.     

Secondary structure of β-hydroxydecanoyl thiol ester dehydrase, a 39-kDa protein, derived from Hα, Cα, Cβ and CO signal assignments and the Chemical Shift Index: Comparison with the crystal structure

Summary Nearly complete backbone ¹H, ¹⁵N and ¹³C signal assignments are reported for -hydroxydecanoyl thiol ester dehydrase, a 39-kDa homodimer containing 342 amino acids. Although ¹⁵N relaxation data show that the protein has a rotational correlation time of 18 ns, assignments were derived from triple-resonance experiments recorded at 500 MHz and pH 6.8, without deuteration. The Chemical Shift Index, CSI, identified two long helices and numerous -strands in dehydrase. The CSI predictions are in close agreement with the secondary structure identified in the recently derived crystal structure, particularly when one takes account of the numerous bulges in the -strands. The assignment of dehydrase and a large deuterated protein [Yamazaki et al. (1994) J. Am. Chem. Soc., 116, 11655–11666] suggest that assignment of 40–60 kDa proteins is feasible. Hence, further progress in understanding the chemical shift/structure relationship could open the way to determine the structures of such large proteins. Supplementary Material is available on request, comprising Table S1 listing the spectral parameters; Table S2 listing the assignments; Fig. S1 showing the 2D ¹H–¹⁵N HSQC spectrum; Fig. S2 showing sequential NOEs, secondary shifts, H-exchange and ³J_HN data; and Fig. S3 showing plots of the H, C, CO and C Chemical Shift Indexes.To whom correspondence should be addressed.