Gerald R. Reeck

“Homology” in proteins and nucleic acids: A terminology muddle and a way out of it

“Homology” has the precise meaning in biology of “having a common evolutionary origin,” but it also carries the loose meaning of “possessing similarity or being matched.” Its rampant use in the loose sense is clogging the literature on protein and nucleic acid sequence comparisons with muddy writing and, in some cases, muddy thinking In its precise biological meaning, “homology” is a concept of quality. The word asserts a type of relationship between two or more things. Thus, amino acid or nucleotide sequences are either homologous or they are not. They cannot exhibit a particular “level of homology” or “percent homology.” Instead, two sequences possess a certain level of similarity. Similarity is thus a quantitative property. Homologous proteins or nucleic acid segments can range from highly similar to not recognizably similar (where similarity has disappeared through divergent evolution). If using “homology” loosely did not interfere with our thinking about evolutionary relationships, the way in which we use the term would be a rather unimportant semantic issue. The fact is, however, that loose usage in sequence comparison papers often makes it difficult to know the authors intent and can lead to confusion for the reader (and even for the author). There are three common situations in which hazards arise by using “homology” to mean similarity. The first case is the most obvious offense but perhaps the least troublesome. Here an author identifies sequence similarities (calling them homologies) but claims that the sequences being compared are not evolutionarily related. Some awkward moments occur in such a paper, since the author claims both homology (i.e., similarity) and nonhomology (i.e., lack of a common ancestor). Nonetheless, the author’s ideas are likely to be clear since arguments against common ancestry are presented explicitly. A second case is one in which an author points out similarities (again called homologies) but does not address the issue of evolutionary origins. The reader, seeing the term “homology,” may infer that the author is postulating coancestry when that is not the authors intent. The final case occurs most frequently and is the most subtle and therefore most troublesome. Here, similarities (called homologies) are used to support a hypothesis of evolutionary homology. In this case, the two meanings of homology seem to overlap, and it is almost inevitable that the thinking of author and reader alike will be intrusively distorted as follows. Similarity is relatively straightforward to document. In comparing sequences, a similarity can take the form of a numerical score (O/o amino acid or nucleotide positional identity, in the simplest approach) or of a probability associated with such a score. In comparisons of three-dimensional structures, a typical numerical description is root-mean-square positional deviation between compared atomic positions. A similarity, then, can become a fully documented, simple fact. On the other hand, a common evolutionary origin must usually remain a hypothesis, supported by a set of arguments that might include sequence or three-dimensional similarity. Not all similarity connotes homology but that can be easily overlooked if similarities are called homologies. Thus, in this third case, we can deceive ourselves into thinking we have proved something substantial (evolutionary homology) when, in actuality, we have merely established a simple fact (a similarity, mislabeled as homology). Homology among similar structures is a hypothesis that may be correct or mistaken, but a similarity itself is a fact, however it is interpreted. We believe that the concepts of evolutionary homology and sequence or three-dimensional similarity can be kept distinct only if they are referred to with different words. We therefore offer the following recommendations: *Sequence similarities (or other types of similarity) should simply be called similarities. They should be documented by appropriate statistical analysis. In writing about sequence similarities the following sorts of terms might be used: a level or degree of similarity; an alignment with optimized similarity; the % positional identity in an alignment; the probability associated with an alignment. *Homology should mean “possessing a common evolutionary origin” and in the vast majority of reports should have no other meaning. Evidence for evolutionary homology should be explicitly laid out, making it clear that the proposed relationship is based on the level of observed similarity, the statistical significance of the similarity, and possibly other lines of reasoning. One could argue that the meaning of the term “homology” is itself evolving. But if that evolution is toward vagueness and if it results in making our scientific discourse unclear, surely we should intervene. With a collective decision to mend our ways, proper usage would soon become fashionable and therefore easy. We believe that we and our scientific heirs would benefit significantly.

RNAi Knockdown of a Salivary Transcript Leading to Lethality in the Pea Aphid, Acyrthosiphon pisum

Abstract Injection of siRNA (small interfering RNA) into parthenogenetic adult pea aphids (Acyrthosiphon pisum) is shown here to lead to depletion of a target salivary gland transcript. The siRNA was generated from double stranded RNA that covered most of the open reading frame of the transcript, which we have called Coo2. The Coo2 transcript level decreases dramatically over a 3-day period after injection of siRNA. With a lag of 1 to 2 days, the siCoo2-RNA injected insects died, on average 8 days before the death of control insects injected with siRNA for green fluorescent protein. It appears, therefore, that siRNA injections into adults will be a useful tool in studying the roles of individual transcripts in aphid salivary glands and suggests that siCoo2-RNA injections can be a useful positive control in such studies.

A protein from the salivary glands of the pea aphid, Acyrthosiphon pisum, is essential in feeding on a host plant

In feeding, aphids inject saliva into plant tissues, gaining access to phloem sap and eliciting (and sometimes overcoming) plant responses. We are examining the involvement, in this aphid–plant interaction, of individual aphid proteins and enzymes, as identified in a salivary gland cDNA library. Here, we focus on a salivary protein we have arbitrarily designated Protein C002. We have shown, by using RNAi-based transcript knockdown, that this protein is important in the survival of the pea aphid (Acyrthosiphon pisum) on fava bean, a host plant. Here, we further characterize the protein, its transcript, and its gene, and we study the feeding process of knockdown aphids. The encoded protein fails to match any protein outside of the family Aphididae. By using in situ hybridization and immunohistochemistry, the transcript and the protein were localized to a subset of secretory cells in principal salivary glands. Protein C002, whose sequence contains an N-terminal secretion signal, is injected into the host plant during aphid feeding. By using the electrical penetration graph method on c002-knockdown aphids, we find that the knockdown affects several aspects of foraging and feeding, with the result that the c002-knockdown aphids spend very little time in contact with phloem sap in sieve elements. Thus, we infer that Protein C002 is crucial in the feeding of the pea aphid on fava bean.

Predicted effector molecules in the salivary secretome of the pea aphid (Acyrthosiphon pisum): a dual transcriptomic/proteomic approach.

The relationship between aphids and their host plants is thought to be functionally analogous to plant-pathogen interactions. Although virulence effector proteins that mediate plant defenses are well-characterized for pathogens such as bacteria, oomycetes, and nematodes, equivalent molecules in aphids and other phloem-feeders are poorly understood. A dual transcriptomic-proteomic approach was adopted to generate a catalog of candidate effector proteins from the salivary glands of the pea aphid, Acyrthosiphon pisum. Of the 1557 transcript supported and 925 mass spectrometry identified proteins, over 300 proteins were identified with secretion signals, including proteins that had previously been identified directly from the secreted saliva. Almost half of the identified proteins have no homologue outside aphids and are of unknown function. Many of the genes encoding the putative effector proteins appear to be evolving at a faster rate than homologues in other insects, and there is strong evidence that genes with multiple copies in the genome are under positive selection. Many of the candidate aphid effector proteins were previously characterized in typical phytopathogenic organisms (e.g., nematodes and fungi) and our results highlight remarkable similarities in the saliva from plant-feeding nematodes and aphids that may indicate the evolution of common solutions to the plant-parasitic lifestyle.

Inhibition of digestive proteinase of stored grain coleoptera by oryzacystatin, a cysteine proteinase inhibitor from rice seed

Electrophoresis of midgut extracts from the rice weevil, Sithopilus oryzae, and the red flour beetle, Tribolium castaneum, in polyacrylamide gels containing sodium dodecyl sulfate and gelatin revealed there was one major proteinase (apparent molecular mass = 40 000) in the rice weevil and two major proteinase (apparent molecular masses = 20 000 and 17 000) in the red flour beetle. The pH optima using [3PH]casein as substrate were about pH 6.8. for the rice weevil and pH 5.2 for the red flour beetle. Use of specific inhibitors, including L‐trans‐epoxysuccinyl‐leucylamino‐(4‐guanidino)‐butane (E‐64). p‐chloromercuriphenylsulfonic acid (PCMS), and oryzacystatin, indicated that nearly all of the proteinase activity against casein was contributed by cysteine proteinases. The estimated IC10 values for oryzacystatin were 2 × 10−6 M and 4 × 10−7 M when tested against midgut extracts from T. castaneum and S. oryzae, respectively.

Expression of a cysteine proteinase inhibitor (oryzacystatin-I) in transgenic tobacco plants

Expression of cysteine proteinase inhibitors (cystatins) in tobacco or other plants has the potential for improving resistance against pathogens and insects that possess cysteine proteinases. A chimeric gene containing a cDNA clone of rice cystatin (oryzacystatin-I; OC-I), the cauliflower mosaic virus 35S promoter, and the nopaline synthase 3′ region was introduced into tobacco plants by Agrobacterium tumefaciens. The presence of the chimeric gene in transgenic plants was detected by a polymerase chain reaction-amplified assay, and transcriptional activity was shown by RNA blot analysis. Heated extracts from transgenic tobacco plants, as well as from progeny which were obtained by selfing a primary transformant, contained protein bands that corresponded in molecular mass to OC-I and reacted with antibodies raised against rOC, a recombinant OC-I protein produced by Escherichia coli. Similar bands were absent in extracts from untransformed control plants. OC-I levels reached 0.5% and 0.6% of the total soluble proteins in leaves and roots, respectively, of some progeny. On a fresh weight basis, the OC-I content was higher in leaves (50 μg/g) than in roots (30 μg/g). OC-I was partially purified from protein extracts of rice seeds and from transgenic tobacco leaves by affinity to anti-rOC antibodies. OC-I from both sources was active against papain.

Rice cystatin: bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein.

A cDNA clone that encodes oryzacystatin, a cysteine protease inhibitor from rice, was isolated and expressed in Escherichia coli BL-21 (DE3) using an expression plasmid under the control of a T7 RNA polymerase promoter. The construct pT7OC 9b encoded a fusion protein containing 11 amino acid residues of the NH2 terminus of the bacterial protein phi 10 and 79 residues of oryzacystatin lacking 23 NH2-terminal residues of the wild-type protein. Recombinant oryzacystatin (ROC) constituted approximately 10% of the total bacterial protein mass and was purified in a single step by anion-exchange chromatography. The inhibitory activity of ROC toward papain (Ki = 3 x 10(-8) M) was comparable with that of the naturally occurring protein isolated from rice. Caseinolytic activity in midgut homogenates from seven species of stored product insects was inhibited from 18 to 85% by ROC, whereas the same activity was inhibited from 14 to 69% by the serine proteinase inhibitor phenylmethylsulfonyl fluoride. Midguts of stored product insects apparently contain both cysteine proteinases and serine proteinases, but the relative amounts vary with the species. When fed to the red flour beetle, Tribolium castaneum, 10 wt% ROC in the diet suppressed growth approximately 35% relative to that of the control group of insects.

α-amylase inhibitors from wheat: Amino acid sequences and patterns of inhibition of insect and human α-amylases

Four alpha-amylase inhibitors, WRP24, WRP25, WRP26, and WRP27, were purified from wheat flour by preparative, reversed-phase high performance liquid chromatography. All have polypeptide molecular masses of about 14 kDa and are members of the cereal superfamily of protease and alpha-amylase inhibitors. Sedimentation velocity analysis indicated that WRP25 and WRP27 are monomeric proteins, whereas WRP24 is a dimer. WRP24 is identical in N-terminal amino acid sequence to the well characterized 0.19 dimeric inhibitor from wheat kernels. WRP25 and WRP26 differ in sequence from each other at only three positions and represent previously unseparated forms of the 0.28 wheat inhibitor. WRP27 is a previously uncharacterized inhibitor and is more similar in sequence to the 0.28 inhibitor than to the 0.19 inhibitor. WRP25 and WRP26 inhibited alpha-amylases from the rice weevil, red flour beetle, and the yellow meal worm, but did not inhibit human salivary alpha-amylase. WRP24 inhibited the human as well as the insect alpha-amylases, but inhibited one of the two rice weevil alpha-amylases much more strongly than the other. WRP27 was notable in that, of the enzymes tested, it strongly inhibited only the rice weevil alpha-amylases. We observed that the growth rate of red flour beetle larvae was slowed when purified WRP24 was included in the diet at a level of 10%. Addition of WRP24 to corn starch resulted in greater weight loss of red flour beetle adults than occurred on control diets. Our results support the hypothesis that these alpha-amylase inhibitors provide wheat seeds with a selective evolutionary advantage since the inhibitors can slow the growth of insect pests that attack cereal grains.

Nucleotide sequence of a rice genomic clone that encodes a class I endochitinase

TTGCTGAAGATCGATGCACCATGCA TATCCATCTCTATATAAAGCCATGCGATCCCACCGATTCTTGCACACACACTAGCTACTT CTACTTCTATCATACCAAACAAACTAGCTTAATTTGCATTGCATCACATTGCCGGCCGCC ATGAGAGCTCTCGCTCTCGCGGTGGTGGCCATGGCGGTGGTGGCCGTGCGCGGCGAGCAG M R A L A L A V V A M A V V A V R G E Q TGCGGCAGCCAGGCCGGCGGCGCGCTCTGCCCCAACTGCCTCTGCTGCAGCCAGTACGGC C G S Q A G G A L C P N C L C C S Q Y G TGGTGCGGCTCCACCTCCGATTACTGCGGCGCCGGCTGCCAGAGCCAGTGCTCCGGCGGC W C G S T S D Y C G A G C Q S Q C S G G TGCGGCGGCGGCCCGACCCCGCCCTCCAGCGGTGGCGGCAGCGGCGTCGCCTCCATCATA C G G G P T P P S S G G G S G V A S I I TCGCCCTCGCTCTTCGACCAGATGCTGCTCCACCGCAACGACCAGGCGTGCCGCGCTAAG S P S L F D Q M L L H R N D Q A C R A K GGCTTCTACACCTACGACGCCTTCGTCGCCGCCGCCAACGCCTACCCGGACTTCGCCACC G F Y T Y D A F V A A A N A Y P D F A T ACGCGCGACGCCGACACCTGCAAGCGCGAGGTCGCCGCCTTCCTGGCGCAGACGTCCCAC T R D A D T C K R E V A A F L A Q T S H GAGACCACCGGCGGCTGGCCCACGGCGCCCGACGGCCCCTACTCCTGGGGCTACTGCTTC E T T G G W P T A P D G P Y S W G Y C F AAGGAGGAGAACAACGGCAACGCCCCCACATACTGCGAGCCCAAGCCGGAGTGGCCGTGC K E E N N G N A P T Y C E P K P E W P C GCCGCCGCGAAGAAGTACTACGGCCGGGGACCCATCCAGATCACCTACAACTACAACTAC A A A K K Y Y G R G P I Q I T Y N Y N Y GGCCGCGGGGCAGGCATCGGCTCCGACCTGCTCAACAACCCGGACCTGGTGGCGTCGGAC G R G A G I G S D L L N N P D L V A S D GCAGTCTCCTTCAAGACGGCGTTCTGGTTCTGGATGACGCCGCAGTCGCCCAAGCCGTCG A V S F K T A F W F W M T P Q S P K P S TGCCACGCGGTGATCACCGGCCAGTGGACGCCGTCCGCCGACGACCAGGCGGCGGGGCGC C H A V I T G Q W T P S A D D Q A A G R GTTCCGGGCTACGGCGAGATCACCAACATCATCAACGGCGGTGTGGAGTGCGGGCACGGC V P G Y G E I T N I I N G G V E C G H G GCGGACGACAAGGTGGCCGACCGGATCGGGTTCTACAAGCGCTACTGCGACATGCTGGGC A D D K V A D R I G F Y K R Y C D M L G GTCAGCTATGGCGATAACCTGGATTGCTACAACCAGAGGCCCTACCCGCCTTCCTAGTTG V S Y G D N L D C Y N Q R P Y P P S < ATATTTGATCCGAGCAGACGAATAAAATACAATGCACACGAGATTGTGAGACTCGTGAAA AACATATACTACCTCTGAATTTTAATACATATCTCTAAAACAAAGATTTGATCCGTTGAC CTGCAGGTCGAC 1237 Fig. 1.

Photoluminescent graphene nanoparticles for cancer phototherapy and imaging.

Graphene-based nanomaterials are of great interest in a wide range of applications in electronics, the environment, and energy as well as in biomedical and bioengineering. Their unique properties make them generally applicable as prognostic, diagnostic, and therapeutic agents in cancer. In this work, we focused on photodynamic and photothermal therapeutic properties of our previously synthesized carboxylated photoluminescent graphene nanodots (cGdots). The cGdots are ∼5 nm in diameter and excited at 655 nm. Our findings reveal that, upon laser irradiation by near-infrared (wavelength 670 nm) sensitizer, electrons of the cGdots starts to vibrate and form electron clouds, thereby generating sufficient heat (>50 °C) to kill the cancer cells by thermal ablation. The generation of singlet oxygen also occurs due to irradiation, thus acting similarly to pheophorbide-A, a well-known photodynamic therapeutic agent. The cGdots kills MDA-MB231 cancer cells (more than 70%) through both photodynamic and photothermal effects. The cGdots were equally effective in the in vivo model of MDA-MB231 xenografted tumor-bearing mice also as observed for 21 days. The cGdot was intravenously injected, and the tumor was irradiated by laser, resulting in final volume of tumor was ∼70% smaller than that of saline-treated tumor. It indicates that the growth rate of cGdot-treated tumor was slower compared to saline-treated tumor. The synthesized cGdots could enable visualization of tumor tissue in mice, thereby illustrating their use as optical imaging agents for detecting cancer noninvasively in deep tissue/organ. Collectively, our findings reveal that multimodal cGdots can be used for phototherapy, through photothermal or photodynamic effects, and for noninvasive optical imaging of deep tissues and tumors simultaneously.