Discovery Talk Questions and Answers: Discovering Programmed Cell Death

H. Robert Horvitz
Assessments created by Dr. Rachna Sharma

Questions

In the paper, the authors mention that they found “that heat-shocked hsp-bcl-2 transgenic animals had almost twice as many neurons in the ventral cord as heat-shocked animals transgenic for a hsp-vector construct or as non-heat-shocked animals.”
What did this result suggest? (Select all that apply)
1. That expression of bcl-2 can restore the function of ced-9 (lf) mutants.
2. That bcl-2 is involved in the cell death pathway.
3. That bcl-2 can potentiate Ced-9 activity.
4. All of the above.
From the list below, choose a likely outcome of an experiment resulting in the overexpression of bcl-2 in hsp-bcl-2 transgenic C. elegans.
1. The number of cells will increase during C. elegans development.
2. The number of cells will decrease during C. elegans development.
3. The organism will die during development.
4. None of the above
In the paper, the expression of ced-9 is referred to as a “bicistronic.”
Does this mean that:
1. Ced-9 has two cistrons, spliced later into single cistronic transcripts.
2. Ced-9 is transcribed together with another gene adjacent to it as part of a single transcript.
3. None of the above
In the paper, the authors mention that the sequence homology between ced-9 and bcl-2 proteins is 89%.
Do you agree or disagree with the following statement?
‘This high level of sequence homology is what gives them the ability to replace each other functionally.’ Explain why you agree or disagree.
In his talk, Dr. Horovitz refers to his student, Michael Hengartner, who was trying to characterize the ced-9 gene, which ultimately led to the groundbreaking discovery of similarities in genetic pathways between organisms.
1. What important observation did Dr. Hengartner make that led to this discovery?
2. Why was this observation so groundbreaking?
In the paper, the authors describe how the ced-9 gene was cloned from genomic DNA: they first identified large DNA fragments that could possibly contain the ced-9 gene.
What strategy did they use to narrow down the fragments that could possibly contain ced-9?
The functional ability of a gene is further characterized by its ability to encode for RNA transcripts and proteins.
In the paper, what was the method used to identify the correct ced-9 ORF and the protein encoded by it?
In the cell paper the authors indicate that ced-9 and bcl-2 are homologous. They however found only 23% similarity in sequence between the two genes.
What are the other probable ways to further analyze the sequences and look for functional similarity?
Why is programmed cell death is important at all stages of life from embryogenesis to aging. What would happen if cell death were completely inhibited in an organism?

Answers

1. a, b, cp. 672: “These results indicate that bcl-2 can prevent the ectopic cell deaths that occur in ced-9(lf) mutants and therefore that bcl-2 interacts with the cell death pathway either at the same step or downstream of ced-9, supporting the hypothesis that bcl-2 can substitute for ced-9. However, we cannot formally exclude the alternative possibility that bcl-2 acted in this experiment by potentiating residual Ced-9 activity contributed maternally or zygotically.”
a.
Bcl-2 protects cells from undergoing apoptosis in C. elegans. Ced-9 shows significant sequence and structural similarity to mammalian bcl-2. Over expression of bcl-2 prevents the normal cell deaths that occur during C. elegans development and increases the cell number.p. 671: “bcl-2 Prevents the Ectopic Cell Deaths in ced-9(lf) mutants: The observation that bcl-2 can prevent the normal programmed cell deaths that occur during C. elegans development indicates that bcl-2 can interact with the C. elegans cell death pathway (…). The sequence similarity between ced-9 and bcl-2 suggests that bcl-2 acts at the same step as ced-9 normally acts, i.e., that bcl-2 mimics the effect of ced-9 on C. elegans programmed cell death. If so, then bcl-2 might be able to substitute for ced-9.”
b.
p. 669: “regulation of ced-9 expression : detection of bicistronic transcripts containing both ORFs, predicts that at least some ced-9 transcripts arise from a 2.1 kb bicistronic message that is trans-spliced at the ced-9 5’ end to the SL1 splice leader to generate the mature cyt-7 and ced-9 transcripts (Figure 6).”p. 672: “ced-9 appears to be an element of a complex locus that includes the gene cyt-1, which encodes a C. elegans cytochrome b560 homolog. The existence of a cyt-1-ced-9 bicistronic message and the requirement for sequences upstream of cyt-1 for ced-9 expression suggest that at least some ced-9 mRNA arises from bicistronic message that is processed by trans-splicing.”
This statement is correct: the function of bcl-2 and ced-9 is equivalent.
Note tha, the goal of this question is to encourage students to formulate a complete opinion, and justify their answer:p. 669: “Interestingly, the proposed function of the bcl-2 oncogene is equivalent to the function we proposed for ced-9 (Hengartner et al., 1992): bcl-2 is thought to be required to prevent cells from undergoing programmed cell death (reviewed by Korsmeyer, 1992). These similarities in sequence and biological function suggest that bcl-2 could be a vertebrate homolog of ced-9. The low overall degree of similarity between ced-9 and bcl-2 prompted us to seek further evidence that these two proteins are homologous. We found that ced-9 and bcl-2 share a number of additional features.”
a. Michael finds that the ced-9 gene shares significant similarity with a human gene, which is a human cancer gene – a proto-oncogene referred to as bcl-2. Like ced-9, this gene was known to protect cells from programmed cell death.Video (04:56): “I was at a scientific conference, and Michael had been studying one of the genes that we had characterized in our analyses of C. elegans programmed cell death, a gene called ced-9. Ced for cell death abnormal, gene number 9. And ced-9 was a key gene in the regulation of programmed cell death in C. elegans. And what Michael was trying to do was to characterize this gene, not just through formal genetic analysis, but also through molecular analysis. And the first step in this process was to identify a molecular clone of ced-9 and look and see that it reminded us of any other gene that was known. What Michael’s fax told me was that when he had searched the literature, and this was very early days of gene sequencing of this sort… When he has searched the database and looked to see if there were any similar genes out there, one emerged at the top of the list, far above anything else. And this match was a human gene. It was a human cancer gene–a proto-oncogene known as Bcl2. Now, ced-9 had been shown to protect cells in C. elegans from programmed cell death–our studies. Bcl2, from work of cancer biologists, had been shown to protect cells against programmed cell death, and to cause cancer because it was protecting cells from dying that normally should die. So, cells that should die instead lived. That led to their survival, and consequently led to cancerous growth. So, this finding that a worm gene that protects against programmed cell death during C. elegans development and a human gene that protects against programmed cell death, and when misexpressed basically would protect cells that should die from doing so, thereby leading to cancer. This finding said that these two genes that function similarly look similar in their sequence. And, this was the finding that said to me that if these two genes are so similar in both function and structure, there must be a pathway of genes that is similar between organisms as diverse as this microscopic ground worm and us.”b. This discovery was key for the field: it demonstrated that results obtained in C. elegans would probably be applied to other organisms and vice versa.
Video (8:00): “This was a moment of excitement. I was absolutely thrilled because what it said was that the studies we had been doing in terms of analysis of C. elegans were going to be relevant to an understanding of human biology and human disease. I should add that it was this finding that made the biomedical community pay attention. Prior to this, I was basically doing abstract genetic studies of an organism most people were paying no attention to involving a phenomenon that most people were paying no attention to. Suddenly, we were working on a gene and a pathway that was key in human disease. Our work was no longer abstractions from genetics, but suddenly had a strong foothold in the future of human biology. And I would say the rest is history.”
p. 666: “Mapping ced-9 with respect to these RFLPs localized the gene to a roughly 60kb interval between the RFLPs nP55 and nP56 (Figure 1b). (…) individual cosmids were used to generate transgenic lines through germline transformation and transgenic ced-9(lf) animals were tested for fertility and viability. Three overlapping cosmids were found to be able to rescue the ced-9 mutant phenotype (figure 1c).”The authors then further narrowed down the potential region by looking at the common region between these cosmids.
The goal of the question is to emphasize the strategies used, such as frameshift mutations, deletions, rescuing activity of ced-9 mutants used to assess function of the mutants.p. 667: “To determine which of the two identified ORFs corresponds to the ced-9 gene, we introduced frameshift mutations into each ORF and tested the resulting constructs for their abilities to rescue the sterility and lethality of ced-9(lf) mutants.”p. 668: “To confirm these observations, we also determined the sequences of several ced-9 mutants. We found changes in the downstream ORF in three distinct ced-9(lf) mutants (Figure 4). For example, the strong ced-9 mutation n2077, which we previously identified as a potential null allele (Hengartner et al., 1992), was associated with a C to T transition at position 2668, resulting in a premature amber stop codon at position 160 of the downstream ORF. Consistent with this observation, we have found that the amber suppressor mutation sup-7(st5)X weakly suppressed the sterility (but not the lethality) caused by ced-9(n7950 n2077) mutations (M. 0. H. and H. Ft. H., unpublished data). These results strongly suggest that ced-9 corresponds to the downstream ORF, which can encode a protein of 260 amino acids and about 32 kd.”
In figure 7, the protein sequences from various species are aligned and show conserved or highly similar regions in important functional and/or structural domains.p. 669: “The low overall degree of similarity between ced-9 and bcl-2 prompted us to seek further evidence that these two proteins are homologous. We found that ced-9 and bcl-2 share a number of additional features. First, the C-terminal halves of the two proteins show similar Kyte-Doolittle hydrophobicity plots (data not shown), including the hydrophobic tail (Figure 7a), thought to be important for bcl-2 localization, function, or both (Alnemri et al., 1992; Tanaka et al., 1992; Hockenbery et al., 1993). Second, the last ced-9 intron is in the same position of the ORF as are the last introns of the human, mouse, and chicken bcl-2genes (Negrini et al., 1987; Seto et al., 1988; Eguchi et al., 1992), as well as the human bcl-x (Boise et al., 1993) and bax (Oltvai et al., 1993) genes (Figure 7a).Also refer to p. 670: “Conserved Regions between CED-9 and N-2”
Programmed cell death is required at all developmental stages of an organism from embryogenesis to an adult to maintain homeostasis or balance. It is essential for some cells to die during development, and prevention of their death can lead to chronic diseases like cancer, autoimmune disorders and several neurological disorders. Development is a very precise mechanism and is a balance between cell division, cell differentiation and programmed cell death.